Subtilase variants

ABSTRACT

The present invention relates to subtilase variants having a reduced tendency towards inhibition by substances present in eggs, such as trypsin inhibitor type IV-0. In particular, the variants comprise at least one additional amino acid residue between positions 42-43, 51-56, 155-161, 187-190, 216-217, 217-218 or 218-219 (in BASBPN numbering). These subtilase variants are useful exhibiting excellent or improved wash performance on egg stains when used in e.g. cleaning or detergent compositions, such as laundry detergent compositions and dishwash composition, including automatic dishwash compositions. Also, isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention. Further, cleaning and detergent compositions comprising the variants are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims, under 35 U.S.C. 119, priority or thebenefit of Danish application no. PA 2000 01528 filed Oct. 13, 2000 andU.S. provisional application No. 60/241,201 filed Oct. 17, 2000, thecontents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to subtilase variants having areduced tendency towards inhibition by substances present in eggs, suchas trypsin inhibitor type IV-0. In particular, the present inventionrelates to subtilase variants comprising at least one additional aminoacid residue between positions 42-43, 51-55, 155-160, 187-189, 217-218or 218-219 (in subtilisin BPN′ (BASBPN) numbering, vide infra). Thesesubtilase variants are useful exhibiting excellent or improved washperformance on egg stains when used in e.g. cleaning or detergentcompositions, such as laundry detergent compositions and dishwashcomposition, including automatic dishwash compositions. The presentinvention also relates to isolated DNA sequences encoding the variants,expression vectors, host cells, and methods for producing and using thevariants of the invention. Further, the present invention relates tocleaning and detergent compositions comprising the variants of theinvention.

BACKGROUND OF THE INVENTION

[0003] In the detergent industry enzymes have for more than 30 yearsbeen implemented in washing formulations. Enzymes used in suchformulations comprise proteases, lipases, amylases, cellulases, as wellas other enzymes, or mixtures thereof. Commercially most importantenzymes are proteases.

[0004] An increasing number of commercially used proteases are proteinengineered variants of naturally occurring wild type proteases, e.g.DURAZYM® (Novozymes A/S), RELASE® (Novozymes A/S), MAXAPEM® PURAFECT®(Genencor International, Inc.).

[0005] Further, a number of protease variants are described in the art.A thorough list of prior art protease variants is given in WO 99/27082.

[0006] However, even though a number of useful proteases and proteasevariants have been described, there is still a need for new improvedproteases or protease variants for a number of industrial uses.

[0007] In particular, the problem of removing egg stains from e.g.laundry or hard surfaces has been pronounced due to the fact thatsubstances present in the egg white inhibit many serine proteases.Examples of such substances include trypsin inhibitor type IV-0(Ovo-inhibitor) and trypsin inhibitor type III-0 (Ovomucoid).

[0008] Therefore, an object of the present invention is to provideimproved subtilase enzymes, which are not, or which are only to alimited extent, inhibited by such substances. A further object of thepresent invention is to provide improved subtilase enzymes that aresuitable for removal of egg stains from, for example, laundry and/orhard surfaces.

SUMMARY OF THE INVENTION

[0009] Thus, in a first aspect the present invention relates to asubtilase variant selected from the group consisting of

[0010] (a) a subtilase variant comprising an insertion of at least oneadditional amino acid residue between positions 42 and 43 (BASBPNnumbering);

[0011] (b) a subtilase variant comprising an insertion of at least oneadditional amino acid residue between positions 51 and 56 (BASBPNnumbering);

[0012] (c) a subtilase variant comprising an insertion of at least oneadditional amino acid residue between positions 155 and 161 (BASBPNnumbering);

[0013] (d) a subtilase variant comprising an insertion of at least oneadditional amino acid residue between positions 187 and 190 (BASBPNnumbering);

[0014] (e) a subtilase variant comprising an insertion of at least oneadditional amino acid residue between positions 216 and 217 (BASBPNnumbering); and

[0015] (f) a subtilase variant comprising an insertion of at least oneadditional amino acid residue between positions 217 and 218 (BASBPNnumbering); and

[0016] (g) a subtilase variant comprising an insertion of at least oneadditional amino acid residue between positions 218 and 219 (BASBPNnumbering).

[0017] In a second aspect the present invention relates to an isolatednucleic acid sequence comprising a nucleic acid sequence that encodesfor a subtilase variant according to the invention.

[0018] In a third aspect the present invention relates to a nucleic acidconstruct comprising the nucleic acid sequence according to theinvention operably linked to one or more control sequences capable ofdirecting the expression of the subtilase in a suitable host.

[0019] In a fourth aspect the present invention relates to a recombinantexpression vector comprising the nucleic acid construct according to theinvention, a promoter, and transcriptional and translational stopsignals.

[0020] In a fifth aspect the present invention relates to a recombinanthost cell comprising the nucleic acid construct of the invention.

[0021] In a sixth aspect the present invention relates to a method forproducing a subtilase variant according to the invention, the methodcomprising:

[0022] (a) cultivating a recombinant host cell according to theinvention under conditions conducive to the production of the subtilase;and

[0023] (b) recovering the subtilase variant.

[0024] In a seventh aspect the present invention relates to a method forproducing a subtilase variant according to the invention, the methodcomprising:

[0025] (a) cultivating a strain from the genus Bacillus, preferably fromthe species Bacillus clausii, such as Bacillus clausii DSM 13585, toproduce a supernatant comprising the subtilase variant; and

[0026] (b) recovering the subtilase variant.

[0027] In an eight aspect the present invention relates to a cleaning ordetergent composition, preferably a laundry or dishwash composition,comprising a subtilase variant according to the invention.

[0028] Further aspects of the present invention relate to use of thesubtilases according to the invention in a cleaning or detergentcomposition; use of the subtilases or the compositions according to theinvention for removal of egg stains; a method for cleaning or washing,including a method for removal of egg stains from, a hard surface orlaundry comprising contacting the hard surface or the laundry with thecomposition of the invention.

[0029] In a seventh aspect the present invention relates to a method forremoval of egg stains from a hard surface or from laundry, the methodcomprising contacting the egg stain-containing hard surface or the eggstain-containing laundry with a cleaning or detergent composition,preferably a laundry or dishwash composition, which contains a subtilasevariant according to the invention.

[0030] Concerning alignment and numbering reference is made to FIG. 1which shows an alignments between subtilisin BPN′ (a) (BASBPN) (SEQ IDNO: 7) and subtilisin 309 (b) (BLSAVI) (SEQ ID NO: 8).

[0031] These alignments are in this patent application used as areference for numbering the residues.

DEFINITIONS

[0032] Prior to discussing this invention in further detail, thefollowing terms and conventions will first be defined.

[0033] Nomenclature of Amino Acids and Nucleic Acids

[0034] Throughout the specification, figures and claims the recognizedIUPAC nomenclature for amino acid residues will be used, mostly in theone letter code form, but also in the three letter code form. Similarlyrecognized IUPAC nomenclature for nucleic acids will be used throughoutthe specification, figures and claims.

[0035] Nomenclature and Conventions For Designation of Variants

[0036] In describing the various subtilase enzyme variants produced orcontemplated according to the invention, the following nomenclatures andconventions have been adapted for ease of reference:

[0037] A frame of reference is first defined by aligning the isolated orparent enzyme with subtilisin BPN′ (BASBPN).

[0038] The alignment can be obtained by the GAP routine of the GCGpackage version 9.1 to number the variants using the followingparameters: gap creation penalty=8 and gap extension penalty=8 and allother parameters kept at their default values.

[0039] Another method is to use known recognized alignments betweensubtilases, such as the alignment indicated in WO 91/00345. In mostcases the differences will not be of any importance.

[0040] Thereby a number of deletions and insertions will be defined inrelation to BASBPN. In FIG. 1, subtilisin 309 has 6 deletions inpositions 36, 58, 158, 162, 163, and 164 in comparison to BASBPN. Thesedeletions are in FIG. 1 indicated by asterixes (*).

[0041] The various modifications performed in a parent enzyme isindicated in general using three elements as follows:

[0042] Original Amino Acid Position Substituted Amino Acid

[0043] The notation G195E thus means a substitution of a glycine inposition 195 with a glutamic acid.

[0044] In the case where the original amino acid residue may be anyamino acid residue, a short hand notation may at times be usedindicating only the position and substituted amino acid:

[0045] Position Substituted Amino Acid

[0046] Such a notation is particular relevant in connection withmodification(s) in homologous subtilases (vide infra).

[0047] Similarly when the identity of the substituting amino acidresidue(s) is immaterial:

[0048] Original Amino Acid Position

[0049] When both the original amino acid(s) and substituted aminoacid(s) may comprise any amino acid, then only the position isindicated, e.g.: 170.

[0050] When the original amino acid(s) and/or substituted amino acid(s)may comprise more than one, but not all amino acid(s), then the selectedamino acids are indicated inside brackets:

[0051] Original Amino Acid Position {Substituted Amino Acid₁, . . . ,Substituted Amino Acid_(n)}

[0052] For specific variants the specific three or one letter codes areused, including the codes Xaa and X to indicate any amino acid residue.

[0053] Substitutions:

[0054] The substitution of glutamic acid for glycine in position 195 isdesignated as:

[0055] Gly195Glu or G195E

[0056] or the substitution of any amino acid residue acid for glycine inposition 195 is designated as:

[0057] Gly195Xaa or G195X

[0058] or

[0059] Gly195 or G195

[0060] The substitution of serine for any amino acid residue in position170 would thus be designated

[0061] Xaa170Ser or X170S

[0062] or

[0063] 170Ser or 170S

[0064] Such a notation is particular relevant in connection withmodification(s) in homologous subtilases (vide infra). 170Ser is thusmeant to comprise e.g. both a Lys170Ser modification in BASBPN andArg170Ser modification in BLSAVI (cf. FIG. 1).

[0065] For a modification where the original amino acid(s) and/orsubstituted amino acid(s) may comprise more than one, but not all aminoacid(s), the substitution of glycine, alanine, serine or threonine forarginine in position 170 would be indicated by

[0066] Arg170{Gly,Ala,Ser,Thr} or R170{G,A,S,T}

[0067] to indicate the variants

[0068] R170G, R170A, R170S, and R170T.

[0069] Deletions:

[0070] A deletion of glycine in position 195 will be indicated by:

[0071] Gly195* or G195*

[0072] Correspondingly the deletion of more than one amino acid residue,such as the deletion of glycine and leucine in positions 195 and 196will be designated

[0073] Gly195*+Leu196* or G195*+L196*

[0074] Insertions:

[0075] The insertion of an additional amino acid residue such as e.g. alysine after G195 is indicated by:

[0076] Gly195GlyLys or G195GK;

[0077] or, when more than one amino acid residue is inserted, such ase.g. a Lys, and Ala after G195 this will be indicated as:

[0078] Gly195GlyLysAla or G195GKA

[0079] In such cases the inserted amino acid residue(s) are numbered bythe addition of lower case letters to the position number of the aminoacid residue preceding the inserted amino acid residue(s). In the aboveexample the sequences 194 to 196 would thus be: 194 195 196 ELSAVI A -G - L 194 195 195a 195b 196 Variant A - G - K -  A -  L (SEQ ID NO: 1)

[0080] In cases where an amino acid residue identical to the existingamino acid residue is inserted it is clear that a degeneracy in thenomenclature arises. If for example a glycine is inserted after theglycine in the above example this would be indicated by G195GG. The sameactual change could just as well be indicated as A194AG for the changefrom 194 195 196 BLSAVI A - G - L to 194 195  195a 196 Variant A - G-  G -  L (SEQ ID NO: 2) 194 194a 195  196

[0081] Such instances will be apparent to the skilled person, and theindication G195GG and corresponding indications for this type ofinsertions are thus meant to comprise such equivalent degenerateindications.

[0082] Filling a Gap:

[0083] Where a deletion in an enzyme exists in the reference comparisonwith the subtilisin BPN′ sequence used for the numbering, an insertionin such a position is indicated as:

[0084] *36Asp or *36D

[0085] for the insertion of an aspartic acid in position 36.

[0086] Multiple Modifications:

[0087] Variants comprising multiple modifications are separated bypluses, e.g.:

[0088] Arg170Tyr+Gly195Glu or R170Y+G195E

[0089] representing modifications in positions 170 and 195 substitutingtyrosine and glutamic acid for arginine and glycine, respectively.

[0090] Thus, Tyr167{Gly,Ala,Ser,Thr}+Arg170{Gly,Ala,Ser,Thr} designatesthe following variants: Tyr167Gly + Arg170Gly, Tyr167Gly + Arg170Ala,Tyr167Gly + Arg170Ser, Tyr167Gly + Arg170Thr, Tyr167Ala + Arg170Gly,Tyr167Ala + Arg170Ala, Tyr167Ala + Arg170Ser, Tyr167Ala + Arg170Thr,Tyr167Ser + Arg170Gly, Tyr167Ser + Arg170Ala, Tyr167Ser + Arg170Ser,Tyr167Ser + Arg170Thr, Tyr167Thr + Arg170Gly, Tyr167Thr + Arg170Ala,Tyr167Thr + Arg170Ser, and Tyr167Thr + Arg170Thr.

[0091] This nomenclature is particular relevant relating tomodifications aimed at substituting, replacing, inserting or deletingamino acid residues having specific common properties, such as residuesof positive charge (K, R, H), negative charge (D, E), or conservativeamino acid modification(s) of e.g.Tyr167{Gly,Ala,Ser,Thr}+Arg170{Gly,Ala,Ser,Thr}, which signifiessubstituting a small amino acid for another small amino acid. Seesection “Detailed description of the invention” for further details.

[0092] Proteases

[0093] Enzymes cleaving the amide linkages in protein substrates areclassified as proteases, or (interchangeably) peptidases (see Walsh,1979, Enzymatic Reaction Mechanisms. W.H. Freeman and Company, SanFrancisco, Chapter 3).

[0094] Numbering of Amino Acid Positions/Residues

[0095] If nothing else is mentioned the amino acid numbering used hereincorrespond to that of the subtilase BPN′ (BASBPN) sequence. For furtherdescription of the BPN′ sequence, see FIG. 1 or Siezen et al., ProteinEngng. 4 (1991) 719-737.

[0096] Serine Proteases

[0097] A serine protease is an enzyme which catalyzes the hydrolysis ofpeptide bonds, and in which there is an essential serine residue at theactive site (White, Handler and Smith, 1973 “Principles ofBiochemistry,” Fifth Edition, McGraw-Hill Book Company, NY, pp.271-272).

[0098] The bacterial serine proteases have molecular weights in the20,000 to 45,000 Dalton range. They are inhibited bydiisopropylfluorophosphate. They hydrolyze simple terminal esters andare similar in activity to eukaryotic chymotrypsin, also a serineprotease. A more narrow term, alkaline protease, covering a subgroup,reflects the high pH optimum of some of the serine proteases, from pH9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41711-753).

[0099] Subtilases

[0100] A sub-group of the serine proteases tentatively designatedsubtilases has been proposed by Siezen et al., Protein Engng. 4 (1991)719-737 and Siezen et al. Protein Science 6 (1997) 501-523. They aredefined by homology analysis of more than 170 amino acid sequences ofserine proteases previously referred to as subtilisin-like proteases. Asubtilisin was previously often defined as a serine protease produced byGram-positive bacteria or fungi, and according to Siezen et al. now is asubgroup of the subtilases. A wide variety of subtilases have beenidentified, and the amino acid sequence of a number of subtilases hasbeen determined. For a more detailed description of such subtilases andtheir amino acid sequences reference is made to Siezen et al. (1997).

[0101] One subgroup of the subtilases, I-S1 or “true” subtilisins,comprises the “classical” subtilisins, such as subtilisin 168 (BSS168),subtilisin BPN′ (BASBPN), subtilisin Carlsberg (BLSCAR) (ALCALASE®,Novozymes A/S), and subtilisin DY (BSSDY). Other subgroup I-S1subtilases of interest are subtilisins closely related to BSS168, suchas subtilisin Amylosacchariticus (BSSAS), subtilisin J (BSAPRJ),subtilisin NAT (BSAPRN), and mesentericopeptidase (BMSAMP); subtilisinsclosely related to BLSCAR, such as Keratinase (BLKERA), subtilisinCarlsberg 11594 (BLSCA1), subtilisin Carlsberg 15413 (BLSCA2),subtilisin Carlsberg 14353 (BLSCA3); and the subtilisins serine proteaseC (BSSPRC), and serine protease D (BSSPRD), or functional variantsthereof having retained the characteristic of sub-group I-S1.

[0102] A further subgroup of the subtilases, I-S2 or high alkalinesubtilisins, is recognized by Siezen et al. (supra). Sub-group I-S2proteases are described as highly alkaline subtilisins and comprisesenzymes such as subtilisin PB92 (BAALKP) (MAXACAL®, Gist-Brocades NV),subtilisin 309 (BLSAVI, BLS309)(SAVINASE®, Novozymes A/S), subtilisin147 (BLS147) (ESPERASE®, Novozymes A/S), and alkaline elastase YaB(BSEYAB). Other subgroup I-S2 subtilases of interest are subtilisin ALPI (BSAPRQ), subtilisins closely related to BLS147, such as subtilisinAprM (BSAPRM), and alkaline protease AH-101 (BAH101)), SAVINASE (BLSAVI)(or the closely related subtilisins M-protease (BSKSMK), subtilisin PB92(BAALKP), and subtilisin BL (BLSUBL)), alkaline elastase YaB (BSEYAB),Thermitase (TVTHER), and subtilisin Sendai (BSAPRS), or functionalvariants thereof having retained the characteristic of sub-group I-S2.

[0103] “Savinase®”

[0104] SAVINASE® is marketed by NOVOZYMES A/S. It is subtilisin 309 fromB. Lentus and differs from BAALKP only in one position (N87S, see FIG. 1herein). SAVINASE® has the amino acid sequence designated b) in FIG. 1.

[0105] Parent Subtilase

[0106] The term “parent subtilase” describes a subtilase definedaccording to Siezen et al. (1991 and 1997). For further details seedescription of “SUBTILASES” immediately above. A parent subtilase mayalso be a subtilase isolated from a natural source, wherein subsequentmodifications have been made while retaining the characteristic of asubtilase. Furthermore, a parent subtilase may also be a subtilase whichhas been prepared by the DNA shuffling technique, such as described byJ. E. Ness et al., Nature Biotechnology, 17, 893-896 (1999).Alternatively the term “parent subtilase” may be termed “wild typesubtilase”.

[0107] Modification(s) of a Subtilase Variant

[0108] The term “modification(s)” used herein is defined to includechemical modification of a subtilase as well as genetic manipulation ofthe DNA encoding a subtilase. The modification(s) can be replacement(s)of the amino acid side chain(s), substitution(s), deletion(s) and/orinsertions in or at the amino acid(s) of interest.

[0109] Subtilase Variant

[0110] In the context of this invention, the term subtilase variant ormutated subtilase means a subtilase that has been produced by anorganism which is expressing a mutant or modified gene derived from aparent microorganism which possessed an original or parent gene andwhich produced a corresponding parent enzyme, the parent gene havingbeen mutated in order to produce the mutant gene from which said mutatedsubtilase protease is produced when expressed in a suitable host.Analogously, the mutant gene may also be derived from a parent geneproduced by DNA shuffling technique.

[0111] Homologous Subtilase Sequences

[0112] The homology between two amino acid sequences is in this contextdescribed by the parameter “identity”.

[0113] In order to determine the degree of identity between twosubtilases the GAP routine of the GCG package version 9.1 can be applied(infra) using the same settings. The output from the routine is besidesthe amino acid alignment the calculation of the “Percent Identity”between the two sequences.

[0114] Based on this description it is routine for a person skilled inthe art to identify suitable homologous subtilases and correspondinghomologous active site loop regions, which can be modified according tothe invention.

[0115] Isolated DNA Sequence or Polynucleotide

[0116] The term “isolated nucleic acid sequence” as used herein refersto a polynucleotide molecule with a defined nucleic acid sequence, whichhas been isolated and purified and is thus in a form suitable for usewithin genetically engineered protein production systems. Such isolatedmolecules may be those that are separated from their natural environmentand include cDNA and genomic clones as well as polynucleotides ornucleic acid sequences derived from DNA shuffling experiments or fromsite-directed mutagenesis experiments. Isolated polynucleotides ornucleic acid sequences of the present invention are free of other geneswith which they are ordinarily associated, but may include 5′ and 3′untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985). The term “isolated nucleic acid sequence” or “isolatedpolynucleotides” may alternatively be termed “isolated DNA sequence,“cloned polynucleotide”, “cloned nucleic acid sequence” or “cloned DNAsequence”.

[0117] Isolated Protein

[0118] When applied to a protein, the term “isolated” indicates that theprotein has been removed from its native environment.

[0119] In a preferred form, the isolated protein is substantially freeof other proteins, particularly other homologous proteins (i.e.“homologous impurities” (see below)).

[0120] An isolated protein is more than 10% pure, preferably more than20% pure, more preferably more than 30% pure, as determined by SDS-PAGE.Further it is preferred to provide the protein in a highly purifiedform, i.e., more than 40% pure, more than 60% pure, more than 80% pure,more preferably more than 95% pure, and most preferably more than 99%pure, as determined by SDS-PAGE.

[0121] The term “isolated protein” may alternatively be termed “purifiedprotein”.

[0122] Homologous Impurities

[0123] The term “homologous impurities” means any impurity (e.g. anotherpolypeptide than the subtilase of the invention), which originate fromthe homologous cell where the subtilase of the invention is originallyobtained from.

[0124] Obtained From

[0125] The term “obtained from” as used herein in connection with aspecific microbial source, means that the polynucleotide and/orsubtilase produced by the specific source, or by a cell in which a genefrom the source has been inserted.

[0126] Substrate

[0127] The term “substrate” used in connection with a substrate for aprotease should be interpreted in its broadest form as comprising acompound containing at least one peptide (amide) bond susceptible tohydrolysis by a subtilisin protease.

[0128] Product

[0129] The term “product” used in connection with a product derived froma protease enzymatic reaction should, in the context of the presentinvention, be interpreted to include the products of a hydrolysisreaction involving a subtilase protease. A product may be the substratein a subsequent hydrolysis reaction.

[0130] Wash Performance

[0131] In the present context the term “wash performance” is used as anenzyme's ability to remove egg stains present on the object to becleaned during e.g. wash or hard surface cleaning. See also the “ModelDetergent Wash Performance Test” in Example 3 herein.

[0132] Performance Factor

[0133] The term “Performance Factor” is defined with respect to thebelow formula

P=R _(variant) −R _(parent)

[0134] wherein P is the Performance Factor, R_(variant) is thereflectance (measured at 460 nm) of the test material after beingtreated with a subtilase variant as described in the “Model DetergentWash Performance Test”, and R_(parent) is the reflectance (measured at460 nm) of the test material after being treated with the correspondingparent subtilase as described in the “Model Detergent Wash PerformanceTest”. For further details, see the “Model Detergent Wash PerformanceTest” in Example 3 herein.

[0135] Residual Activity

[0136] The term “Residual Activity” is defined as described in the“Ovo-inhibition Assay” herein (see Example 3).

BRIEF DESCRIPTION OF THE DRAWING

[0137]FIG. 1 shows an alignment between subtilisin BPN′ (a) andSAVINASE® (b) using the GAP routine mentioned above.

DETAILED DESCRIPTION OF THE INVENTION

[0138] The present inventors have found that subtilisin variants,wherein certain regions are longer than those presently known, areinhibited by substances present in eggs, such as trypsin inhibitor typeIV-0, to a significantly lesser extent than the parent subtilase and,consequently, the variants according to the invention exhibit improvedwash performance with respect to removal of egg stains.

[0139] The identification thereof was done in constructing subtilisinvariants, especially of the subtilisin 309 (BLSAVI or SAVINASE®).Without being limited to any specific theory it is presently believedthat due to steric hindrance and/or conformational changes, binding ofthe egg white inhibitor in the substrate binding region of the subtilasevariant is impeded.

[0140] Thus, variants which are contemplated as being suitable for theuses described herein are such variants where, when compared to thewild-type subtilase, one or more amino acid residues has been insertedin one or more of the following positions: between positions 42 and 43,between positions 51 and 52, between positions 52 and 53, betweenpositions 53 and 54, between positions 54 and 55, between positions 55and 56, between positions 155 and 156, between positions 156 and 157,between positions 157 and 158, between positions 158 and 159, betweenpositions 159 and 160, between positions 160 and 161, between positions187 and 188, between positions 188 and 189, between positions 189 and190, between positions 216 and 217, between positions 217 and 218, orbetween positions 218 and 219 (BASBPN numbering), in particular betweenpositions 217 and 218.

[0141] A subtilase variant of the first aspect of the invention may be aparent or wild-type subtilase identified and isolated from nature.

[0142] Such a parent wildtype subtilase may be specifically screened forby standard techniques known in the art.

[0143] One preferred way of doing this may be by specifically PCRamplify DNA regions known to encode active site loops in subtilases fromnumerous different microorganism, preferably different Bacillus strains.

[0144] Subtilases are a group of conserved enzymes, in the sense thattheir DNA and amino acid sequences are homologous. Accordingly it ispossible to construct relatively specific primers flanking active siteloops.

[0145] One way of doing this is by investigating an alignment ofdifferent subtilases (see e.g. Siezen et al. Protein Science 6 (1997)501-523). It is from this routine work for a person skilled in the artto construct PCR primers flanking the positions indicated above in anyof the group I-S1 or I-S2 groups, such as from BLSAVI. Using such PCRprimers to amplify DNA from a number of different microorganism,preferably different Bacillus strains, followed by DNA sequencing ofsaid amplified PCR fragments, it will be possible to identify strainswhich produce subtilases of these groups comprising insertions, ascompared to e.g. BLSAVI sequence. Having identified the strain and apartial DNA sequence of such a subtilase of interest, it is routine workfor a person skilled in the art to complete cloning, expression andpurification of such a subtilase.

[0146] However, it is envisaged that a subtilase variant of theinvention predominantly is a variant of a parent subtilase.

[0147] A subtilase variant suitable for the uses described herein, maybe constructed by standard techniques known in the art such as bysite-directed/random mutagenesis or by DNA shuffling of differentsubtilase sequences. See the “Material and Methods” section herein (videinfra) for further details.

[0148] As will be acknowledged by the skilled person, the variantsdescribed herein may comprise one or more further modifications, inparticular one or more further insertions or substitutions.

[0149] Moreover, the insertions in the regions described herein mayencompass insertion of more than just one amino acid residue. Forexample the variant according to the invention may contain oneinsertion, two insertions, or more than two insertions, such as three,four or five insertions.

[0150] In one interesting embodiment of the invention the additionalamino acid residue is inserted between positions 42 and 43.

[0151] The insertion between positions 42 and 43 is preferably selectedfrom the group consisting of (in BASBPN numbering)

[0152] X42X{A,T,G,S}, e.g., X42XA, X42XT, X42XG, X42XS;

[0153] X42X{D,E,K,R}, e.g., X42XD, X42XE, X42XK, X42XR;

[0154] X42X{H,V,C,N,Q}, e.g., X42XH, X42XV, X42XC, X42XN, X42XQ; and

[0155] X42X{F,I,L,M,P,W,Y}, e.g., X42XF, X42XI, X42XL, X42XM, X42XP,X42XW, X42XY;

[0156] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0157] D42D{A,T,G,S}, e.g., D42DA, D42DT, D42DG, D42DS;

[0158] D42D{D,E,K,R}, e.g., D42DD, D42DE, D42DK, D42DR;

[0159] D42D{H,V,C,N,Q}, e.g., D42DH, D42DV, D42DC, D42DN, D42DQ; and

[0160] D42D{F,I,L,M,P,W,Y}, e.g., D42DF, D42DI, D42DL, D42DM, D42DP,D42DW, D42DY.

[0161] In a further interesting embodiment of the invention theadditional amino acid residue is inserted between positions 51 and 52.

[0162] The insertion between positions 51 and 52 is preferably selectedfrom the group consisting of (in BASBPN numbering)

[0163] X51X{A,T,G,S}, e.g., X51XA, X51XT, X51XG, X51XS;

[0164] X51X{D,E,K,R}, e.g., X51XD, X51XE, X51XK, X51XR;

[0165] X51X{H,V,C,N,Q}, e.g., X51XH, X51XV, X51XC, X51XN, X51XQ; and

[0166] X51X{F,I,L,M,P,W,Y}, e.g., X51XF, X51XI, X51XL, X51XM, X51XP,X5lXW, X5lXY;

[0167] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0168] V51V{A,T,G,S}, e.g., V51VA, V51VT, V51VG, V51VS;

[0169] V51V{D,E,K,R}, e.g., V51VD, V51VE, V51VK, V51VR;

[0170] V51V{H,V,C,N,Q}, e.g., V51VH, V51VV, V51VC, V51VN, V51VQ; and

[0171] V51V{F,I,L,M,P,W,Y}, e.g., V51VF, V51VI, V51VL, V51VM, V51VP,V5lVW, V5lVY.

[0172] In another interesting embodiment of the invention the additionalamino acid residue is inserted between positions 52 and 53.

[0173] The insertion between positions 52 and 53 is preferably selectedfrom the group consisting of (in BASBPN numbering)

[0174] X52X{A,T,G,S}, e.g., X52XA, X52XT, X52XG, X52XS;

[0175] X52X{D,E,K,R}, e.g., X52XD, X52XE, X52XK, X52XR;

[0176] X52X{H,V,C,N,Q}, e.g., X52XH, X52XV, X52XC, X52XN, X52XQ; and

[0177] X52X{F,I,L,M,P,W,Y}, e.g., X52XF, X52XI, X52XL, X52XM, X52XP,X52XW, X52XY;

[0178] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0179] P52P{A,T,G,S}, e.g., P52PA, P52PT, P52PG, P52PS;

[0180] P52P{D,E,K,R}, e.g., P52PD, P52PE, P52PK, P52PR;

[0181] P52P{H,V,C,N,Q}, e.g., P52PH, P52PV, P52PC, P52PN, P52PQ; and

[0182] P52P{F,I,L,M,P,W,Y}, e.g., P52PF, P52PI, P52PL, P52PM, P52PP,P52PW, P52PY.

[0183] In further interesting embodiment of the invention the additionalamino acid residue is inserted between positions 53 and 54.

[0184] The insertion between positions 53 and 54 is preferably selectedfrom the group consisting of (in BASBPN numbering)

[0185] X53X{A,T,G,S}, e.g., X53XA, X53XT, X53XG, X53XS;

[0186] X53X{D,E,K,R}, e.g., X53XD, X53XE, X53XK, X53XR;

[0187] X53X{H,V,C,N,Q}, e.g., X53XH, X53XV, X53XC, X53XN, X53XQ; and

[0188] X53X{F,I,L,M,P,W,Y}, e.g., X53XF, X53XI, X53XL, X53XM, X53XP,X53XW, X53XY;

[0189] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0190] G53G{A,T,G,S}, e.g., G53GA, G53GT, G53GG, G53GS;

[0191] G53G{D,E,K,R}, e.g., G53GD, G53GE, G53GK, G53GR;

[0192] G53G{H,V,C,N,Q}, e.g., G53GH, G53GV, G53GC, G53GN, G53GQ; and

[0193] G53G{F,I,L,M,P,W,Y}, e.g., G53GF, G53GI, G53GL, G53GM, G53GP,G53GW, G53GY.

[0194] In a still further interesting embodiment of the invention theadditional amino acid residue is inserted between positions 54 and 55.

[0195] The insertion between positions 54 and 55 is preferably selectedfrom the group consisting of (in BASBPN numbering)

[0196] X54X{A,T,G,S}, e.g., X54XA, X54XT, X54XG, X54XS;

[0197] X54X{D,E,K,R}, e.g., X54XD, X54XE, X54XK, X54XR;

[0198] X54X{H,V,C,N,Q}, e.g., X54XH, X54XV, X54XC, X54XN, X54XQ; and

[0199] X54X{F,I,L,M,P,W,Y}, e.g., X54XF, X54XI, X54XL, X54XM, X54XP,X54XW, X54XY;

[0200] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0201] E54E{A,T,G,S}, e.g., E54EA, E54ET, E54EG, E54ES;

[0202] E54E{D,E,K,R}, e.g., E54ED, E54EE, E54EK, E54ER;

[0203] E54E{H,V,C,N,Q}, e.g., E54EH, E54EV, E54EC, E54EN, E54EQ; and

[0204] E54E{F,I,L,M,P,W,Y}, e.g., E54EF, E54EI, E54EL, E54EM, E54EP,E54EW, E54EY.

[0205] In an even further interesting embodiment of the invention theadditional amino acid residue is inserted between positions 55 and 56.

[0206] The insertion between positions 55 and 56 is preferably selectedfrom the group consisting of (in BASBPN numbering)

[0207] X55X{A,T,G,S}, e.g., X55XA, X55XT, X55XG, X55XS;

[0208] X55X{D,E,K,R}, e.g., X55XD, X55XE, X55XK, X55XR;

[0209] X55X{H,V,C,N,Q}, e.g., X55XH, X55XV, X55XC, X55XN, X55XQ; and

[0210] X55X{F,I,L,M,P,W,Y}, e.g., X55XF, X55XI, X55XL, X55XM, X55XP,X55XW, X55XY;

[0211] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0212] P55P{A,T,G,S}, e.g., P55PA, P55PT, P55PG, P55PS;

[0213] P55P{D,E,K,R}, e.g., P55PD, P55PE, P55PK, P55PR;

[0214] P55P{H,V,C,N,Q}, e.g., P55PH, P55PV, P55PC, P55PN, P55PQ; and

[0215] P55P{F,I,L,M,P,W,Y}, e.g., P55PF, P55PI, P55PL, P55PM, P55PP,P55PW, P55PY.

[0216] In another interesting embodiment of the invention the additionalamino acid residue is inserted between positions 155 and 156.

[0217] The insertion between positions 155 and 156 is preferablyselected from the group consisting of (in BASBPN numbering)

[0218] X155X{A,T,G,S}, e.g., X155XA, X155XT, X155XG, X155XS;

[0219] X155X{D,E,K,R}, e.g., X155XD, X155XE, X155XK, X155XR;

[0220] X155X{H,V,C,N,Q}, e.g., X155XH, X155XV, X155XC, X155XN, X155XQ;and

[0221] X155X{F,I,L,M,P,W,Y}, e.g., X155XF, X155XI, X155XL, X155XM,X155XP, X155XW, X155XY;

[0222] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0223] N155N{A,T,G,S}, e.g., N155NA, N155NT, N155NG, N155NS;

[0224] N155N{D,E,K,R}, e.g., N155ND, N155NE, N155NK, N155NR;

[0225] N155N{H,V,C,N,Q}, e.g., N155NH, N155NV, N155NC, N155NN, N155NQ;and

[0226] N155N{F,I,L,M,P,W,Y}, e.g., N155NF, N155NI, N155NL, N155NM,N155NP, N155NW, N155NY.

[0227] In a further interesting embodiment of the invention theadditional amino acid residue is inserted between positions 156 and 157.

[0228] The insertion between positions 156 and 157 is preferablyselected from the group consisting of (in BASBPN numbering)

[0229] X156X{A,T,G,S}, e.g., X156XA, X156XT, X156XG, X156XS;

[0230] X156X{D,E,K,R}, e.g., X156XD, X156XE, X156XK, X156XR;

[0231] X156X{H,V,C,N,Q}, e.g., X156XH, X156XV, X156XC, X156XN, X156XQ;and

[0232] X156X{F,I,L,M,P,W,Y}, e.g., X156XF, X156XI, X156XL, X156XM,X156XP, X156XW, X156XY;

[0233] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0234] S156S{A,T,G,S}, e.g., S156SA, S156ST, S156SG, S156SS;

[0235] S156S{D,E,K,R}, e.g., S156SD, S156SE, S156SK, S156SR;

[0236] S156S{H,V,C,N,Q}, e.g., S156SH, S156SV, S156SC, S156SN, S156SQ;and

[0237] S156S{F,I,L,M,P,W,Y}, e.g., S156SF, S156SI, S156SL, S156SM,S156SP, S156SW, S156SY.

[0238] In a still further interesting embodiment of the invention theadditional amino acid residue is inserted between positions 157 and 158.

[0239] The insertion between positions 157 and 158 is preferablyselected from the group consisting of (in BASBPN numbering)

[0240] X157X{A,T,G,S}, e.g., X157XA, X157XT, X157XG, X157XS;

[0241] X157X{D,E,K,R}, e.g., X157XD, X157XE, X157XK, X157XR;

[0242] X157X{H,V,C,N,Q}, e.g., X157XH, X157XV, X157XC, X157XN, X157XQ;and

[0243] X157X{F,I,L,M,P,W,Y}, e.g., X157XF, X157XI, X157XL, X157XM,X157XP, X157XW, X157XY;

[0244] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0245] G157G{A,T,G,S}, e.g., G157GA, G157GT, G157GG, G157GS;

[0246] G157G{D,E,K,R), e.g., G157GD, G157GE, G157GK, G157GR;

[0247] G157G{H,V,C,N,Q}, e.g., G157GH, G157GV, G157GC, G157GN, G157GQ;and

[0248] G157G(F,I,L,M,P,W,Y}, e.g., G157GF, G157GI, G157GL, G157GM,G157GP, G157GW, G157GY.

[0249] In an even further interesting embodiment of the invention theadditional amino acid residue is inserted between positions 158 and 159.

[0250] The insertion between positions 158 and 159 is preferablyselected from the group consisting of (in BASBPN numbering)

[0251] X158X{A,T,G,S}, e.g., X158XA, X158XT, X158XG, X158XS;

[0252] X158X{D,E,K,R}, e.g., X158XD, X158XE, X158XK, X158XR;

[0253] X158X{H,V,C,N,Q}, e.g., X158XH, X158XV, X158XC, X158XN, X158XQ;and

[0254] X158X{F,I,L,M,P,W,Y}, e.g., X158XF, X158XI, X158XL, X158XM,X158XP, X158XW, X158XY;

[0255] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0256] *158{A,T,G,S}, e.g., *158A, *158T, *158G, *158S;

[0257] *158{D,E,K,R}, e.g., *158D, *158E, *158K, *158R;

[0258] *158{H,V,C,N,Q}, e.g., *158H, *158V, *158C, *158N, *158Q; and

[0259] *158{F,I,L,M,P,W,Y}, e.g., *158F, *518I, *158L, *158M, *158P,*158W, *158Y.

[0260] In still another interesting embodiment of the invention theadditional amino acid residue is inserted between positions 159 and 160.

[0261] The insertion between positions 159 and 160 is preferablyselected from the group consisting of (in BASBPN numbering)

[0262] X159X{A,T,G,S}, e.g., X159XA, X159XT, X159XG, X159XS;

[0263] X159X{D,E,K,R}, e.g., X159XD, X159XE, X159XK, X159XR;

[0264] X159X{H,V,C,N,Q}, e.g., X159XH, X159XV, X159XC, X159XN, X159XQ;and

[0265] X159X{F,I,L,M,P,W,Y}, e.g., X159XF, X159XI, X159XL, X159XM,X159XP, X159XW, X159XY;

[0266] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0267] A159A{A,T,G,S}, e.g., A159AA, A159AT, A159AG, A159AS;

[0268] A159A{D,E,K,R}, e.g., A159AD, A159AE, A159AK, A159AR;

[0269] A159A{H,V,C,N,Q}, e.g., A159AH, A159AV, A159AC, A159AN, A159AQ;and

[0270] A159A{F,I,L,M,P,W,Y}, e.g., A159AF, A159AI, A159AL, A159AM,A159AP, A159AW, A159AY.

[0271] In a still another interesting embodiment of the invention theadditional amino acid residue is inserted between positions 160 and 161.

[0272] The insertion between positions 160 and 161 is preferablyselected from the group consisting of (in BASBPN numbering)

[0273] X160X{A,T,G,S}, e.g., X160XA, X160XT, X160XG, X160XS;X160X{D,E,K,R}, e.g., X160XD, X160XE, X160XK, X160XR;

[0274] X160X{H,V,C,N,Q}, e.g., X160XH, X160XV, X160XC, X160XN, X160XQ;and

[0275] X160X{F,I,L,M,P,W,Y}, e.g., X160XF, X160XI, X160XL, X160XM,X160XP, X160XW, X160XY;

[0276] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0277] G160G{A,T,G,S}, e.g., G160GA, G160GT, G160GG, G160GS;

[0278] G160G{D,E,K,R}, e.g., G160GD, G160GE, G160GK, G160GR;

[0279] G160G{H,V,C,N,Q}, e.g., G160GH, G160GV, G160GC, G160GN, G160GQ;and

[0280] G160G{F,I,L,M,P,W,Y}, e.g., G160GF, G160GI, G160GL, G160GM,G160GP, G160GW, G160GY.

[0281] In another interesting embodiment of the invention the additionalamino acid residue is inserted between positions 187 and 188.

[0282] The insertion between positions 187 and 188 is preferablyselected from the group consisting of (in BASBPN numbering)

[0283] X187X{A,T,G,S}, e.g., X187XA, X187XT, X187XG, X187XS;

[0284] X187X{D,E,K,R}, e.g., X187XD, X187XE, X187XK, X187XR;

[0285] X187X{H,V,C,N,Q}, e.g., X187XH, X187XV, X187XC, X187XN, X187XQ;and

[0286] X187X{F,I,L,M,P,W,Y}, e.g., X187XF, X187XI, X187XL, X187XM,X187XP, X187XW, X187XY;

[0287] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0288] A187A{A,T,G,S}, e.g., A187AA, A187AT, A187AG, A187AS;

[0289] A187A{D,E,K,R}, e.g., A187AD, A187AE, A187AK, A187AR;

[0290] A187A{H,V,C,N,Q}, e.g., A187AH, A187AV, A187AC, A187AN, A187AQ;and

[0291] A187A{F,I,L,M,P,W,Y}, e.g., A187AF, A187AI, A187AL, A187AM,A187AP, A187AW, A187AY.

[0292] In a further interesting embodiment of the invention theadditional amino acid residue is inserted between positions 188 and 189.

[0293] The insertion between positions 188 and 189 is preferablyselected from the group consisting of (in BASBPN numbering)

[0294] X188X{A,T,G,S}, e.g., X188XA, X188XT, X188XG, X188XS;

[0295] X188X{D,E,K,R}, e.g., X188XD, X188XE, X188XK, X188XR;

[0296] X188X{H,V,C,N,Q}, e.g., X188XH, X188XV, X188XC, X188XN, X188XQ;and

[0297] X188X{F,I,L,M,P,W,Y}, e.g., X188XF, X188XI, X188XL, X188XM,X188XP, X188XW, X188XY;

[0298] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0299] S188S{A,T,G,S}, e.g., S188SA, S188ST, S188SG, S188SS;

[0300] S188S{D,E,K,R}, e.g., S188SD, S188SE, S188SK, S188SR;

[0301] S188S{H,V,C,N,Q}, e.g., S188SH, S188SV, S188SC, S188SN, S188SQ;and

[0302] S188S{F,I,L,M,P,W,Y}, e.g., S188SF, S188SI, S188SL, S188SM,S188SP, S188SW, S188SY.

[0303] In a still further interesting embodiment of the invention theadditional amino acid residue is inserted between positions 189 and 190.

[0304] The insertion between positions 189 and 190 is preferablyselected from the group consisting of (in BASBPN numbering)

[0305] X189X{A,T,G,S}, e.g., X189XA, X189XT, X189XG, X189XS;

[0306] X189X{D,E,K,R}, e.g., X189XD, X189XE, X189XK, X189XR;

[0307] X189X{H,V,C,N,Q}, e.g., X189XH, X189XV, X189XC, X189XN, X189XQ;and

[0308] X189X{F,I,L,M,P,W,Y}, e.g., X189XF, X189XI, X189XL, X189XM,X189XP, X189XW, X189XY;

[0309] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0310] F189F{A,T,G,S}, e.g., F189FA, F189FT, F189FG, F189FS;

[0311] F189F{D,E,K,R}, e.g., F189FD, F189FE, F189FK, F189FR;

[0312] F189F{H,V,C,N,Q}, e.g., F189FH, F189FV, F189FC, F189FN, F189FQ;and

[0313] F189F{F,I,L,M,P,W,Y}, e.g., F189FF, F189FI, F189FL, F189FM,F189FP, F189FW, F189FY.

[0314] In another interesting embodiment of the invention the additionalamino acid residue is inserted between positions 216 and 217.

[0315] The insertion between positions 216 and 217 is preferablyselected from the group consisting of (in BASBPN numbering)

[0316] X216X{A,T,G,S}, e.g., X216XA, X216XT, X21GXG, X216XS;

[0317] X216X{D,E,K,R}, e.g., X216XD, X216XE, X216XK, X216XR;

[0318] X216X{H,V,C,N,Q}, e.g., X216XH, X21GXV, X216XC, X216XN, X216XQ;and

[0319] X216X{F,I,L,M,P,W,Y}, e.g., X216XF, X216XI, X216XL, X216XM,X216XP, X216XW, X216XY;

[0320] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0321] S216S{A,T,G,S}, e.g., S216SA, S216ST, S216SG, S216SS;

[0322] S216S{D,E,K,R}, e.g., S216SD, S216SE, S216SK, S216SR;

[0323] S216S{H,V,C,N,Q}, e.g., S216SH, S216SV, S216SC, S216SN, S216SQ;and

[0324] S216S{F,I,L,M,P,W,Y}, e.g., S216SF, S216SI, S216SL, S216SM,S216SP, S216SW, S216SY.

[0325] In another interesting embodiment of the invention the additionalamino acid residue is inserted between positions 217 and 218.

[0326] The insertion between positions 217 and 218 is preferablyselected from the group consisting of (in BASBPN numbering)

[0327] X217X{A,T,G,S}, e.g., X217XA, X217XT, X217XG, X217XS;

[0328] X217X{D,E,K,R}, e.g., X217XD, X217XE, X217XK, X217XR;

[0329] X217X{H,V,C,N,Q}, e.g., X217XH, X217XV, X217XC, X217XN, X217XQ;and

[0330] X217X{F,I,L,M,P,W,Y}, e.g., X217XF, X217XI, X217XL, X217XM,X217XP, X217XW, X217XY;

[0331] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0332] L217L{A,T,G,S}, e.g., L217LA, L217LT, L217LG, L217LS;

[0333] L217L{D,E,K,R}, e.g., L217LD, L217LE, L217LK, L217LR;

[0334] L217L{H,V,C,N,Q}, e.g., L217LH, L217LV, L217LC, L217LN, L217LQ;and

[0335] L217L{F,I,L,M,P,W,Y}, e.g., L217LF, L217LI, L217LL, L217LM,L217LP, L217LW, L217LY.

[0336] In still another interesting embodiment of the invention theadditional amino acid residue is inserted between positions 218 and 219.

[0337] The insertion between positions 218 and 219 is preferablyselected from the group consisting of (in BASBPN numbering)

[0338] X218X{A,T,G,S}, e.g., X218XA, X218XT, X218XG, X218XS;

[0339] X218X{D,E,K,R}, e.g., X218XD, X218XE, X218XK, X218XR;

[0340] X218X{H,V,C,N,Q}, e.g., X218XH, X218XV, X218XC, X218XN, X218XQ;and

[0341] X218X{F,I,L,M,P,W,Y}, e.g., X218XF, X218XI, X218XL, X218XM,X218XP, X218XW, X218XY;

[0342] or more specific for subtilisin 309 and closely relatedsubtilases, such as BAALKP, BLSUBL, and BSKSMK:

[0343] N218N{A,T,G,S}, e.g., N218NA, N218NT, N218NG, N218NS;

[0344] N218N{D,E,K,R}, e.g., N218ND, N218NE, N218NK, N218NR;

[0345] N218N{H,V,C,N,Q}, e.g., N218NH, N218NV, N218NC, N218NN, N218NQ;and

[0346] N218N{F,I,L,M,P,W,Y}, e.g., N218NF, N218NI, N218NL, N218NM,N218NP, N218NW, N218NY.

[0347] Moreover, it is contemplated that, in addition to theabove-mentioned insertions performed in accordance with the invention,insertion of at least one additional amino acid residue in the activesite loop (b) region from position 95 to 103 (BASBPN numbering) willfurther reduce the tendency towards inhibition by trypsin inhibitor typeIV-0. It is envisaged that additional insertions between position 98 and99 and/or insertions between positions 99 and 100 will be particularbeneficial. Examples of such additional insertions are:

[0348] X98X{A,T,G,S}, e.g., X98XA, X98XT, X98XG, X98XS;

[0349] X98X{D,E,K,R}, e.g., X98XD, X98XE, X98XK, X98XR;

[0350] X98X{H,V,C,N,Q}, e.g., X98XH, X98XV, X98XC, X98XN, X98XQ; and

[0351] X98X{F,I,L,M,P,W,Y}, e.g., X98XF, X98XI, X98XL, X98XM, X98XP,X98XW, X98XY; preferably X98XD and X98XE;

[0352] or more specific for subtilisin 309 and closely relatedsubtilases:

[0353] A98A{A,T,G,S}, e.g., A98AA, A98AT, A98AG, A98AS;

[0354] A98A{D,E,K,R}, e.g., A98AD, A98AE, A98AK, A98AR;

[0355] A98A{H,V,C,N,Q}, e.g., A98AH, A98AV, A98AC, A98AN, A98AQ;

[0356] A98A{F,I,L,M,P,W,Y}, e.g., A98AF, A98AI, A98AL, A98AM, A98AP,A98AW, A98AY; preferably A98AD and A98AE.

[0357] Further examples include:

[0358] X99X{A,T,G,S}, e.g., X99XA, X99XT, X99XG, X99XS;

[0359] X99X{D,E,K,R}, e.g., X99XD, X99XE, X99XK, X99XR;

[0360] X99X{H,V,C,N,Q}, e.g., X99XH, X99XV, X99XC, X99XN, X99XQ; and

[0361] X99X{F,I,L,M,P,W,Y}, e.g., X99XF, X99XI, X99XL, X99XM, X99XP,X99XW, X99XY; preferably X99XD and X99XE;

[0362] or more specific for subtilisin 309 and closely relatedsubtilases:

[0363] S99S{A,T,G,S}, e.g., S99SA, S99ST, S99SG, S99SS;

[0364] S99S{D,E,K,R}, e.g., S99SD, S99SE, S99SK, S99SR;

[0365] S99S{H,V,C,N,Q}, e.g., S99SH, S99SV, S99SC, S99SN, S99SQ;

[0366] S99S{F,I,L,M,P,W,Y}, e.g., S99SF, S99SI, S99SL, S99SM, S99SP,S99SW, S99SY; preferably S99SD and S99SE.

[0367] With respect to insertions between position 99 and 100, it ispreferred that the insertion is combined with a substitution in position99. Thus, in addition to the contemplated insertions mentioned above,the following substitutions in position 99 are considered relevant:

[0368] X99{A,T,G,S}, e.g., X99A, X99T, X99G, X99S;

[0369] X99{D,E,K,R}, e.g., X99D, X99E, X99K, X99R;

[0370] X99{H,V,C,N,Q}, e.g., X99H, X99V, X99C, X99N, X99Q, and

[0371] X99{F,I,L,M,P,W,Y} e.g., X99F, X99I, X99L, X99M, X99P, X99W,X99Y;

[0372] or more specific for subtilisin 309 and closely relatedsubtilases:

[0373] S99{A,T,G}, e.g., S99A, S99T, S99G;

[0374] S99{D,E,K,R}, e.g., S99D, S99E, S99K, S99R;

[0375] S99{H,V,C,N,Q}, e.g., S99H, S99V, S99C, S99N, S99Q; and

[0376] S99{F,I,L,M,P,W,Y}, e.g., S99F, S99I, S99L, S99M, S99P, S99W,S99SY.

[0377] In a preferred embodiment the substitution in position 99 isselected from the group consisting of X99{A,T,G,S}, in particular X99A,or more specific for subtilisin 309 and closely related subtilases:S99{A,T,G}, in particular S99A.

[0378] It is well known in the art that a so-called conservativesubstitution of one amino acid residue to a similar amino acid residueis expected to produce only a minor change in the characteristic of theenzyme.

[0379] Table I below list groups of conservative amino acidsubstitutions. TABLE I Conservative amino acid substitutions CommonProperty Amino Acid Basic (positive charge) K = lysine H = histidineAcidic (negative charge) E = glutamic acid D = aspartic acid Polar Q =glutamine N = asparagine Hydrophobic L = leucine I = isoleucine V =valine M = methionine Aromatic F = phenylalanine W = tryptophan Y =tyrosine Small G = glycine A = alanine S = serine T = threonine

[0380] According to this principle subtilase variants comprisingconservative substitutions are expected to exhibit characteristics thatare not drastically different from each other.

[0381] Based on the disclosed and/or exemplified subtilase variantsherein, it is routine work for a person skilled in the art to identifysuitable conservative modification(s) to these variants in order toobtain other subtilase variants exhibiting similarly improvedwash-performance.

[0382] It is preferred that the parent subtilase belongs to thesubgroups I-S1 and I-S2, especially subgroup I-S2, both for isolatingenzymes from nature or from the artificial creation of diversity, andfor designing and producing variants from a parent subtilase.

[0383] In relation to variants from subgroup I-S1, it is preferred toselect a parent subtilase from the group consisting of BSS168 (BSSAS,BSAPRJ, BSAPRN, BMSAMP), BASBPN, BSSDY, BLSCAR (BLKERA, BLSCA1, BLSCA2,BLSCA3), BSSPRC, and BSSPRD, or functional variants thereof havingretained the characteristic of sub-group I-S1.

[0384] In relation to variants from subgroup I-S2 it is preferred toselect a parent subtilase from the group consisting of BSAPRQ, BLS147(BSAPRM, BAH101), BLSAVI (BSKSMK, BAALKP (BAPB92), BLSUBL), BSEYAB,TVTHER, and BSAPRS, or functional variants thereof having retained thecharacteristic of sub-group I-S2.

[0385] In particular, the parent subtilase is BLSAVI (SAVINASE®,NOVOZYMES A/S), and a preferred subtilase variant of the invention isaccordingly a variant of SAVINASE®.

[0386] The present invention also encompasses any of the above mentionedsubtilase variants in combination with any other modification to theamino acid sequence thereof. Especially combinations with othermodifications known in the art to provide improved properties to theenzyme are envisaged. The art describes a number of subtilase variantswith different improved properties and a number of those are mentionedin the “Background of the invention” section herein (vide supra). Thosereferences are disclosed here as references to identify a subtilasevariant, which advantageously can be combined with a subtilase variantdescribed herein.

[0387] Such combinations comprise the positions: 222 (improves oxidationstability), 218 (improves thermal stability), substitutions in theCa-binding sites stabilizing the enzyme, e.g. position 76, and manyother apparent from the prior art.

[0388] In further embodiments a subtilase variant described herein mayadvantageously be combined with one or more modification(s) in any ofthe positions (BASBPN numbering):

[0389] 27, 36, 56, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,120, 123, 129, 131, 132, 133, 143, 159, 167, 170, 192, 194, 206, 217,218, 222, 224, 232, 235, 236, 245, 248, 252 or 274

[0390] Specifically, the following variants of the subtilisins SAVINASE(BLSAVI), BLSUBL, BSKSMK, and BAALKP are considered appropriate forcombination:

[0391] K27R, *36D, T56P, N76D, N87S, A97N, A98AT, A98AS, N99ND, N99NR,N99A, N99T, R101G, P103A, V104A, V104I, V104N, V104Y, D120H, N123S,P129K, P131H, A133P, A133D, A133E, T143K, *159D, *159E, Y167X, Y167A,R170X, R170S, A194P, Q206E, F217R, N218S, M222S, M222A, T224S, A232V,K235L, Q236H, Q245R, N248D, N252K and T274A.

[0392] Of further particular interest are variants of the subtilase ofthe invention, wherein the modifications comprise any of themodifications R101G+V104N, S101G+V104N, S87N+S101G+V104N,K27R+V104Y+N123S+T274A, N76D+V104A, orR101G+P103A+V104I+*159D+A232V+Q236H+Q245R+N248D+N252K; or othercombinations of these modifications (K27R, N76D, R101G, S101G, P103A,V104I, V104N, V104A, V104Y, N123S, *159D, A232V, Q236H, Q245R, N248D,N252K T274A), in combination with any one or more of the modification(s)indicated above or below exhibit improved properties.

[0393] A particular interesting variant is a variant, which, in additionto the insertions according to the invention, contains the followingsubstitutions: S101G+S103A+V104I+G159D+A232V+Q236H+Q245R+N248D+N252K.

[0394] Moreover, subtilase variants of the main aspect(s) of theinvention are preferably combined with one or more modification(s) inany of the positions 129, 131 and 194, preferably as 129K, 131H and 194Pmodifications, and most preferably as P129K, P131H and A194Pmodifications. Any of those modification(s) are expected to provide ahigher expression level of the subtilase variant in the productionthereof.

[0395] As mentioned above, the variants of the invention are onlyinhibited by trypsin inhibitor type IV-0 to a limited extent and,consequently, they exhibit excellent wash performance on egg stains.Therefore, in order to enable the skilled person—at an early stage ofhis development work—to select effective and preferred variants for thispurpose, the present inventors have provided a suitable preliminarytest, which can easily be carried out by the skilled person in order toinitially assess the performance of the variant in question.

[0396] Thus, the “Ovo-inhibition Assay” disclosed in Example 4 hereinmay be employed to initially assess the potential of a selected variant.In other words, the “Ovo-inhibition Assay” may be employed to assesswhether a selected variant will be inhibited, and to what extent, by thetrypsin inhibitor type IV-0. Using this test, the suitability of aselected variant to remove egg stains can be assessed, the rationalebeing that if a selected variant is strongly inhibited by trypsininhibitor type IV-0, it is normally not necessary to carry out furthertest experiments.

[0397] Therefore, a variant which is particular interesting for thepurposes described herein, is a variant which—when tested in the“Ovo-inhibition Assay” described in Example 4 herein—has a ResidualActivity of at least 15%, such as at least 20%, preferably at least 25%,such as at least 30%, more preferably at least 35%. In a particularinteresting embodiment of the invention, the variant has a ResidualActivity of at least 40%, such as at least 45%, e.g. at least 50%,preferably at least 55%, such as at least 60%, more preferably at least65%, such as at least 70%, even more preferably at least 75%, such as atleast 80%, e.g. at least 90%, when tested in the “Ovo-inhibition Assay”described in Example 4 herein.

[0398] Evidently, it is preferred that the variant of the inventionfulfils the above criteria on at least the stated lowest level, morepreferably at the stated intermediate level and most preferably on thestated highest level.

[0399] Alternatively, or in addition to the above-mentioned assay, thesuitability of a selected variant may be tested in the “Model DetergentWash Performance Test” disclosed in Example 3 herein. The “ModelDetergent Wash Perfomance Test” may be employed to assess the ability ofa variant, when incorporated in a standard detergent composition, toremove egg stains from a standard textile as compared to a referencesystem, namely the parent subtilase (incorporated in the same modeldetergent system and tested under identical conditions). Using thistest, the suitability of a selected variant to remove egg stains can beinitially investigated, the rationale being that if a selected variantdoes not show a significant improvement in the test compared to theparent subtilase, it is normally not necessary to carry out further testexperiments.

[0400] Therefore, variants which are particular interesting for thepurposes described herein, are such variants which, when tested in amodel detergent composition comprising

[0401] 6.2% LAS (Nansa 80S)

[0402] 2% Sodium salt of C₁₆-C₁₈ fatty acid

[0403] 4% Non-ionic surfactant (Plurafax LF404)

[0404] 22% Zeolite P

[0405] 10.5% Na₂CO₃

[0406] 4% Na₂Si₂O₅

[0407] 2% Carboxymethylcellulose (CMC)

[0408] 6.8% Acrylate liquid CP5 40%

[0409] 20% Sodium perborate (empirical formula NaBO₂.H₂O₂)

[0410] 0.2% EDTA

[0411] 21% Na₂SO₄

[0412] Water (balance)

[0413] as described in the “Model Detergent Wash Performance Test”herein, shows an improved wash performance on egg stains as compared tothe parent subtilase tested under identical conditions.

[0414] The improvement in the wash performance may be quantified byemploying the so-called “Performance Factor” defined in Example 3,herein.

[0415] In a very interesting embodiment of the invention, the variant ofthe invention, when tested in the “Wash Performance Test” has aPerformance Factor of at least 1, such as at least 1.5, e.g. at least 2,preferably at least 2.5, such as at least 3, e.g. at least 3.5, inparticular at least 4, such as at least 4.5, e.g. at least 5.

[0416] Evidently, it is preferred that the variant of the inventionfulfils the above criteria on at least the stated lowest level, morepreferably at the stated intermediate level and most preferably on thestated highest level.

[0417] Producing a Subtilase Variant

[0418] Many methods for cloning a subtilase and for introducinginsertions into genes (e.g. subtilase genes) are well known in the art,cf. the references cited in the “BACKGROUND OF THE INVENTION” section.

[0419] In general standard procedures for cloning of genes andintroducing insertions (random and/or site directed) into said genes maybe used in order to obtain a subtilase variant of the invention. Forfurther description of suitable techniques reference is made to Examplesherein (vide infra) and (Sambrook et al. (1989) Molecular cloning: Alaboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY;Ausubel, F. M. et al. (eds.) “Current protocols in Molecular Biology”.John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.)“Molecular Biological Methods for Bacillus”. John Wiley and Sons, 1990);and WO 96/34946.

[0420] Further, a subtilase variant may be constructed by standardtechniques for artificial creation of diversity, such as by DNAshuffling of different subtilase genes (WO 95/22625; Stemmer WPC, Nature370:389-91 (1994)). DNA shuffling of e.g. the gene encoding SAVINASE®with one or more partial subtilase sequences identified in nature tocomprise insertion(s)in any of the indicated positions in comparison toBLSAVI (SAVINASE®), will after subsequent screening in the ovo-inhibitorassay, provide subtilase variants suitable for the purposes describedherein.

[0421] Expression Vectors

[0422] A recombinant expression vector comprising a DNA constructencoding the enzyme of the invention may be any vector that mayconveniently be subjected to recombinant DNA procedures.

[0423] The choice of vector will often depend on the host cell intowhich it is to be introduced. Thus, the vector may be an autonomouslyreplicating vector, i.e. a vector that exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g. a plasmid.

[0424] Alternatively, the vector may be one that on introduction into ahost cell is integrated into the host cell genome in part or in itsentirety and replicated together with the chromosome(s) into which ithas been integrated.

[0425] The vector is preferably an expression vector in which thepolynucleotide encoding the enzyme of the invention is operably linkedto additional segments required for transcription of the DNA. Ingeneral, the expression vector is derived from plasmid or viral DNA, ormay contain elements of both. The term, “operably linked” indicates thatthe segments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in a promoter andproceeds through the DNA sequence coding for the enzyme.

[0426] The promoter may be any polynucleotide with a sequence, whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell.

[0427] Examples of suitable promoters for use in bacterial host cellsinclude the promoter of the Bacillus stearothermophilus maltogenicamylase gene, the Bacillus licheniformis alpha-amylase gene, theBacillus amyloliquefaciens alpha-amylase gene, the Bacillus subtilisalkaline protease gene, or the Bacillus pumilus xylosidase gene, or thephage Lambda P_(R) or P_(L) promoters or the E. coli lac, trp or tacpromoters.

[0428] The DNA sequence encoding the enzyme of the invention may also,if necessary, be operably connected to a suitable terminator.

[0429] The recombinant vector of the invention may further comprise apolynucleotide enabling the vector to replicate in the host cell inquestion.

[0430] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, or a geneencoding resistance to e.g. antibiotics like kanamycin, chloramphenicol,erythromycin, tetracycline, spectinomycine, or the like, or resistanceto heavy metals or herbicides.

[0431] To direct an enzyme of the present invention into the secretorypathway of the host cells, a secretory signal sequence (also known as aleader sequence, prepro sequence or pre sequence) may be provided in therecombinant vector. The secretory signal sequence is joined to the DNAsequence encoding the enzyme in the correct reading frame. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe enzyme. The secretory signal sequence may be that normallyassociated with the enzyme or may be from a gene encoding anothersecreted protein.

[0432] The procedures used to ligate the DNA sequences coding for thepresent enzyme, the promoter and optionally the terminator and/orsecretory signal sequence, respectively, or to assemble these sequencesby suitable PCR amplification schemes, and to insert them into suitablevectors containing the information necessary for replication orintegration, are well known to persons skilled in the art (cf., forinstance, Sambrook et al., op.cit.).

[0433] Host Cell

[0434] The polynucleotide with a DNA sequence encoding the presentenzyme and introduced into the host cell may be either homologous orheterologous to the host in question. If homologous to the host cell,i.e. produced by the host cell in nature, it will typically be operablyconnected to another promoter sequence or, if applicable, anothersecretory signal sequence and/or terminator sequence than in its naturalenvironment. The term “homologous” is intended to include a DNA sequenceencoding an enzyme native to the host organism in question. The term“heterologous” is intended to include a DNA sequence not expressed bythe host cell in nature. Thus, the DNA sequence may be from anotherorganism, or it may be a synthetic sequence.

[0435] The host cell into which the DNA construct or the recombinantvector of the invention is introduced may be any cell that is capable ofproducing the present enzyme and includes bacteria, yeast, fungi andhigher eukaryotic cells including plants.

[0436] Examples of bacterial host cells which, on cultivation, arecapable of producing the enzyme of the invention are gram-positivebacteria such as strains of Bacillus, such as strains of B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus, B. megatherium or B. thuringiensis, or strains of Streptomyces,such as S. lividans or S. murinus, or gram-negative bacteria such asEcherichia coli.

[0437] The transformation of the bacteria may be effected by protoplasttransformation, electroporation, conjugation, or by using competentcells in a manner known per se (cf. Sambrook et al., (supra).

[0438] When expressing the enzyme in bacteria such as E. coli, theenzyme may be retained in the cytoplasm, typically as insoluble granules(known as inclusion bodies), or may be directed to the periplasmic spaceby a bacterial secretion sequence. In the former case, the cells arelysed and the granules are recovered and denatured after which theenzyme is refolded by diluting the denaturing agent. In the latter case,the enzyme may be recovered from the periplasmic space by disrupting thecells, e.g. by sonication or osmotic shock, to release the contents ofthe periplasmic space and recovering the enzyme.

[0439] When expressing the enzyme in gram-positive bacteria such asBacillus or Streptomyces strains, the enzyme may be retained in thecytoplasm, or may be directed to the extracellular medium by a bacterialsecretion sequence. In the latter case, the enzyme may be recovered fromthe medium as described below.

[0440] Method For Producing a Subtilase Variant

[0441] The present invention provides a method of producing an isolatedenzyme according to the invention, wherein a suitable host cell, whichhas been transformed with a polynucleotide with a DNA sequence encodingthe enzyme, is cultured under conditions permitting the production ofthe enzyme, and the resulting enzyme is recovered from the culture.

[0442] When an expression vector comprising a DNA sequence encoding theenzyme is transformed into a heterologous host cell it is possible toenable heterologous recombinant production of the enzyme of theinvention.

[0443] Thereby it is possible to make a highly purified subtilasecomposition, characterized in being free from homologous impurities.

[0444] In this context, homologous impurities, means any impurities(e.g. other polypeptides than the enzyme of the invention) thatoriginate from the homologous cell from which the enzyme of theinvention is originally obtained.

[0445] The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed subtilase may conveniently be secreted into the culture mediumand may be recovered therefrom by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulfate, followed by chromatographic procedures such asion exchange chromatography, affinity chromatography, or the like.

[0446] Cleaning and Detergent Compositions

[0447] In general, cleaning and detergent compositions are welldescribed in the art and reference is made to WO 96/34946; WO 97/07202;WO 95/30011 for further description of suitable cleaning and detergentcompositions.

[0448] Furthermore the examples herein demonstrate the improvements inwash performance on egg stains for a number of subtilase variants.

[0449] Detergent Compositions

[0450] The subtilase variant may be added to and thus become a componentof a detergent composition.

[0451] The detergent composition of the invention may for example beformulated as a hand or machine laundry detergent composition includinga laundry additive composition suitable for pre-treatment of stainedfabrics and a rinse added fabric softener composition, or be formulatedas a detergent composition for use in general household hard surfacecleaning operations, or be formulated for hand or machine dishwashingoperations.

[0452] In a specific aspect, the invention provides a detergent additivecomprising a subtilase enzyme of the invention. The detergent additiveas well as the detergent composition may comprise one or more otherenzymes such as another protease, a lipase, a cutinase, an amylase, acarbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, agalactanase, a xylanase, an oxidase, e.g., a laccase, and/or aperoxidase.

[0453] In general the properties of the chosen enzyme(s) should becompatible with the selected detergent, (i.e. pH-optimum, compatibilitywith other enzymatic and non-enzymatic ingredients, etc.), and theenzyme(s) should be present in effective amounts.

[0454] Proteases:

[0455] Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metallo protease, preferably an alkaline microbialprotease or a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g. of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

[0456] Examples of useful proteases are the variants described in WO92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially thevariants with substitutions in one or more of the following positions:27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218,222, 224, 235 and 274.

[0457] Preferred commercially available protease enzymes includeAlcalase™, SAVINASE™, Primase™, Duralase™, Esperase™, and Kannase™(Novozymes A/S), Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™,Purafect OxP™, FN2™, and FN3™ (Genencor International Inc.).

[0458] Lipases:

[0459] Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. fromB. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131,253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422).

[0460] Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

[0461] Preferred commercially available lipase enzymes include Lipolase™and Lipolase Ultra™ (Novozymes A/S).

[0462] Amylases:

[0463] Suitable amylases (α and/or β) include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Amylases include, for example, α-amylases obtained fromBacillus, e.g. a special strain of B. licheniformis, described in moredetail in GB 1,296,839.

[0464] Examples of useful amylases are the variants described in WO94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially thevariants with substitutions in one or more of the following positions:15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208,209, 243, 264, 304, 305, 391, 408, and 444.

[0465] Commercially available amylases are Duramyl™, Termamyl™,Fungamyl™ and BAN™ (Novozymes A/S), Rapidase™ and Purastar™ (fromGenencor International Inc.).

[0466] Cellulases:

[0467] Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. No. 4,435,307, No. 5,648,263, No.5,691,178, No. 5,776,757 and WO 89/09259.

[0468] Especially suitable cellulases are the alkaline or neutralcellulases having co lour care benefits. Examples of such cellulases arecellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO96/29397, WO 98/08940. Other examples are cellulase variants such asthose described in WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046,No. 5,686,593, No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

[0469] Commercially available cellulases include Celluzyme™, andCarezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (GenencorInternational Inc.), and KAC-500(B)™ (Kao Corporation).

[0470] Peroxidases/Oxidases:

[0471] Suitable peroxidases/oxidases include those of plant, bacterialor fungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g. from C. cinereus, and variants thereof as those describedin WO 93/24618, NWO 95/10602, and WO 98/15257.

[0472] Commercially available peroxidases include Guardzyme™ (NovozymesA/S).

[0473] The detergent enzyme(s) may be included in a detergentcomposition by adding separate additives containing one or more enzymes,or by adding a combined additive comprising all of these enzymes. Adetergent additive of the invention, i.e. a separate additive or acombined additive, can be formulated e.g. as a granulate, a liquid, aslurry, etc. Preferred detergent additive formulations are granulates,in particular non-dusting granulates, liquids, in particular stabilizedliquids, or slurries.

[0474] Non-dusting granulates may be produced, e.g., as disclosed inU.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated bymethods known in the art. Examples of waxy coating materials arepoly(ethylene oxide) products (polyethylene glycol, PEG) with mean molarweights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50ethylene oxide units; ethoxylated fatty alcohols in which the alcoholcontains from 12 to 20 carbon atoms and in which there are 15 to 80ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

[0475] The detergent composition of the invention may be in anyconvenient form, e.g., a bar, a tablet, a powder, a granule, a paste ora liquid. A liquid detergent may be aqueous, typically containing up to70% water and 0-30% organic solvent, or non-aqueous.

[0476] The detergent composition typically comprises one or moresurfactants, which may be non-ionic including semi-polar and/or anionicand/or cationic and/or zwitterionic. The surfactants are typicallypresent at a level of from 0.1% to 60% by weight. When included thereinthe detergent will usually contain from about 1% to about 40% of ananionic surfactant such as linear alkylbenzenesulfonate,alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcoholethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methylester, alkyl- or alkenylsuccinic acid or soap.

[0477] When included therein the detergent will usually contain fromabout 0.2% to about 40% of a non-ionic surfactant such as alcoholethoxylate, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acylN-alkyl derivatives of glucosamine (“glucamides”).

[0478] The detergent may contain 0-65% of a detergent builder orcomplexing agent such as zeolite, diphosphate, triphosphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates(e.g. SKS-6 from Hoechst).

[0479] The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

[0480] The detergent may contain a bleaching system that may comprise aH₂O₂ source such as perborate or percarbonate that may be combined witha peracid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of e.g. the amide, imide, or sulfone type.

[0481] The enzyme(s) of the detergent composition of the invention maybe stabilized using conventional stabilizing agents, e.g., a polyol suchas propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in e.g. WO 92/19709and WO 92/19708.

[0482] The detergent may also contain other conventional detergentingredients such as e.g. fabric conditioners including clays, foamboosters, suds suppressors, anti-corrosion agents, soil-suspendingagents, anti-soil redeposition agents, dyes, bactericides, opticalbrighteners, hydrotropes, tarnish inhibitors, or perfumes.

[0483] It is at present contemplated that in the detergent compositionsany enzyme, in particular the enzyme of the invention, may be added inan amount corresponding to 0.01-100 mg of enzyme protein per liter ofwash liquor, preferably 0.05-5 mg of enzyme protein per liter of washliquor, in particular 0.1-1 mg of enzyme protein per liter of washliquor.

[0484] The enzyme of the invention may additionally be incorporated inthe detergent formulations disclosed in WO 97/07202, which is herebyincorporated as reference.

[0485] The invention is described in further detail in the followingexamples, which are not in any way intended to limit the scope of theinvention as claimed.

[0486] In the detergent compositions, the abbreviated componentidentifications have the following meanings:

[0487] LAS: Sodium linear C₁₂ alkyl benzene sulphonate

[0488] TAS: Sodium tallow alkyl sulphate

[0489] XYAS: Sodium C_(1X)-C_(1Y) alkyl sulfate

[0490] SS: Secondary soap surfactant of formula 2-butyl octanoic acid

[0491] 25EY: A C₁₂-C₁₅ predominantly linear primary alcohol condensedwith an average of Y moles of ethylene oxide

[0492] 45EY: A C₁₄-C₁₅ predominantly linear primary alcohol condensedwith an average of Y moles of ethylene oxide

[0493] XYEZS: C_(1X)-C_(1Y) sodium alkyl sulfate condensed with anaverage of Z moles of ethylene oxide per mole

[0494] Nonionic: C₁₃-C₁₅ mixed ethoxylated/propoxylated fatty alcoholwith an average degree of ethoxylation of 3.8 and an average degree ofpropoxylation of 4.5 sold under the tradename Plurafax LF404 by BASFGmbH

[0495] CFAA: C₁₂-C₁₄ alkyl N-methyl glucamide

[0496] TFAA: C₁₆-C₁₈ alkyl N-methyl glucamide

[0497] Silicate: Amorphous Sodium Silicate (SiO₂:Na₂O ratio=2.0)

[0498] NaSKS-6: Crystalline layered silicate of formula δ-Na₂Si₂O₅

[0499] Carbonate: Anhydrous sodium carbonate

[0500] Phosphate: Sodium tripolyphosphate

[0501] MA/AA: Copolymer of 1:4 maleic/acrylic acid, average molecularweight about 80,000

[0502] Polyacrylate: Polyacrylate homopolymer with an average molecularweight of 8,000 sold under the tradename PA30 by BASF Gmbh

[0503] Zeolite A: Hydrated Sodium Aluminosilicate of formulaNa₁₂(AlO₂SiO₂)₁₂.27H₂O having a primary particle size in the range from1 to 10 micrometers

[0504] Citrate: Tri-sodium citrate dihydrate

[0505] Citric: Citric Acid

[0506] Perborate: Anhydrous sodium perborate monohydrate bleach,empirical formula NaBO₂.H₂O₂

[0507] PB4: Anhydrous sodium perborate tetrahydrate

[0508] Percarbonate: Anhydrous sodium percarbonate bleach of empiricalformula 2Na₂CO₃.3H₂O₂

[0509] TAED: Tetraacetyl ethylene diamine

[0510] CMC: Sodium carboxymethyl cellulose

[0511] DETPMP: Diethylene triamine penta (methylene phosphonic acid),marketed by Monsanto under the Tradename Dequest 2060

[0512] PVP: Polyvinylpyrrolidone polymer

[0513] EDDS: Ethylene diamine-N,N′-disuccinic acid, [S,S] isomer in theform of the sodium salt

[0514] Suds 25% paraffin wax Mpt 50° C., 17% hydrophobic silica,

[0515] Suppressor: 58% paraffin oil

[0516] Granular Suds 12% Silicone/silica, 18% stearyl alcohol, 70%

[0517] suppressor: starch in granular form

[0518] Sulphate: Anhydrous sodium sulphate

[0519] HMWPEO: High molecular weight polyethylene oxide

[0520] TAE 25: Tallow alcohol ethoxylate (25)

DETERGENT EXAMPLE I

[0521] A granular fabric cleaning composition in accordance with theinvention may be prepared as follows: Sodium linear C₁₂ alkyl 6.5benzene sulfonate Sodium sulfate 15.0 Zeolite A 26.0 Sodiumnitrilotriacetate 5.0 Enzyme 0.1 PVP 0.5 TAED 3.0 Boric acid 4.0Perborate 18.0 Phenol sulphonate 0.1 Minors up to 100%

DETERGENT EXAMPLE II

[0522] A compact granular fabric cleaning composition (density 800 g/l)in accord with the invention may be prepared as follows: 45AS 8.0 25E3S2.0 25E5 3.0 25E3 3.0 TFAA 2.5 Zeolite A 17.0 NaSKS-6 12.0 Citric acid3.0 Carbonate 7.0 MA/AA 5.0 CMC 0.4 Enzyme 0.1 TAED 6.0 Percarbonate22.0 EDDS 0.3 Granular suds suppressor 3.5 water/minors Up to 100%

DETERGENT EXAMPLE III

[0523] Granular fabric cleaning compositions in accordance with theinvention, which are especially useful in the laundering of colouredfabrics were prepared as follows: LAS 10.7 — TAS 2.4 — TFAA — 4.0 45AS3.1 10.0 45E7 4.0 — 25E3S — 3.0 68E11 1.8 — 25E5 — 8.0 Citrate 15.0 7.0Carbonate — 10.0 Citric acid 2.5 3.0 Zeolite A 32.1 25.0 Na-SKS-6 — 9.0MA/AA 5.0 5.0 DETPMP 0.2 0.8 Enzyme 0.10 0.05 Silicate 2.5 — Sulphate5.2 3.0 PVP 0.5 — Poly (4-vinylpyridine)-N- — 0.2 Oxide/copolymer ofvinyl- imidazole and vinyl- pyrrolidone Perborate 1.0 — Phenol sulfonate0.2 — Water/Minors Up to 100%

DETERGENT EXAMPLE IV

[0524] Granular fabric cleaning compositions in accordance with theinvention which provide “Softening through the wash” capability may beprepared as follows: 45AS — 10.0 LAS 7.6 — 68AS 1.3 — 45E7 4.0 — 25E3 —5.0 Coco-alkyl-dimethyl hydroxy- 1.4 1.0 ethyl ammonium chloride Citrate5.0 3.0 Na-SKS-6 — 11.0 Zeolite A 15.0 15.0 MA/AA 4.0 4.0 DETPMP 0.4 0.4Perborate 15.0 — Percarbonate — 15.0 TAED 5.0 5.0 Smectite clay 10.010.0 HMWPEO — 0.1 Enzyme 0.10 0.05 Silicate 3.0 5.0 Carbonate 10.0 10.0Granular suds suppressor 1.0 4.0 CMC 0.2 0.1 Water/Minors Up to 100%

DETERGENT EXAMPLE V

[0525] Heavy duty liquid fabric cleaning compositions in accordance withthe invention may be prepared as follows: LAS acid form — 25.0 Citricacid 5.0 2.0 25AS acid form 8.0 — 25AE2S acid form 3.0 — 25AE7 8.0 —CFAA 5 — DETPMP 1.0 1.0 Fatty acid 8 — Oleic acid — 1.0 Ethanol 4.0 6.0Propanediol 2.0 6.0 Enzyme 0.10 0.05 Coco-alkyl dimethyl — 3.0 hydroxyethyl ammonium chloride Smectite clay — 5.0 PVP 2.0 — Water / Minors Upto 100%

[0526] Powder automatic dishwash composition I Nonionic surfactant0.4-2.5% Sodium metasilicate  0-20% Sodium disilicate  3-20% Sodiumtriphosphate 20-40% Sodium carbonate  0-20% Sodium perborate 2-9%Tetraacetyl ethylene diamine (TAED) 1-4% Sodium sulphate  5-33% Enzymes0.0001-0.1%   Powder automatic dishwash composition II Nonionicsurfactant (e.g. alcohol 1-2% ethoxylate) Sodium disilicate  2-30%Sodium carbonate 10-50% Sodium phosphonate 0-5% Trisodium citratedihydrate  9-30% Nitrilotrisodium acetate (NTA)  0-20% Sodium perboratemonohydrate  5-10% Tetraacetyl ethylene diamine (TAED) 1-2% Polyacrylatepolymer (e.g. maleic  6-25% acid/acrylic acid copolymer) Enzymes0.0001-0.1%   Perfume 0.1-0.5% Water 5-10 Powder automatic dishwashcomposition III Nonionic surfactant 0.5-2.0% Sodium disilicate 25-40%Sodium citrate 30-55% Sodium carbonate  0-29% Sodium bicarbonate  0-20%Sodium perborate monohydrate  0-15% Tetraacetyl ethylene diamine (TAED)0-6% Maleic acid/acrylic acid copolymer 0-5% Clay 1-3% Polyamino acids 0-20% Sodium polyacrylate 0-8% Enzymes 0.0001-0.1%   Powder automaticdishwash composition IV Nonionic surfactant 1-2% Zeolite MAP 15-42%Sodium disilicate 30-34% Sodium citrate  0-12% Sodium carbonate  0-20%Sodium perborate monohydrate  7-15% Tetraacetyl ethylene diamine (TAED)0-3% Polymer 0-4% Maleic acid/acrylic acid copolymer 0-5% Organicphosphonate 0-4% Clay 1-2% Enzymes 0.0001-0.1%   Sodium sulphate BalancePowder automatic dishwash composition V Nonionic surfactant 1-7% Sodiumdisilicate 18-30% Trisodium citrate 10-24% Sodium carbonate 12-20%Monopersulphate (2 KHSO₅.KHSO₄.K₂SO₄) 15-21% Bleach stabilizer 0.1-2%  Maleic acid/acrylic acid copolymer 0-6% Diethylene triaminepentaacetate,   0-2.5% pentasodium salt Enzymes 0.0001-0.1%   Sodiumsulphate, water Balance Powder and liquid dishwash composition withcleaning surfactant system VI Nonionic surfactant   0-1.5% Octadecyldimethylamine N-oxide dihydrate 0-5% 80:20 wt. C18/C16 blend ofoctadecyl 0-4% dimethylamine N-oxide dihydrate and hexadecyldimethylamine N-oxide dihydrate 70:30 wt. C18/C16 blend of octadecyl bis 0-5%(hydroxyethyl)amine N-oxide anhydrous and hexadecyl bis(hydroxyethyl)amine N-oxide anhydrous C₁₃-C₁₅ alkyl ethoxysulfate withan average  0-10% degree of ethoxylation of 3 C₁₂-C₁₅ alkylethoxysulfate with an average 0-5% degree of ethoxylation of 3 C₁₃-C₁₅ethoxylated alcohol with an average 0-5% degree of ethoxylation of 12 Ablend of C₁₂-C₁₅ ethoxylated alcohols with   0-6.5% an average degree ofethoxylation of 9 A blend of C₁₃-C₁₅ ethoxylated alcohols with 0-4% anaverage degree of ethoxylation of 30 Sodium disilicate  0-33% Sodiumtripolyphosphate  0-46% Sodium citrate  0-28% Citric acid  0-29% Sodiumcarbonate  0-20% Sodium perborate monohydrate   0-11.5% Tetraacetylethylene diamine (TAED) 0-4% Maleic acid/acrylic acid copolymer   0-7.5%Sodium sulphate   0-12.5% Enzymes 0.0001-0.1%  

[0527] Non-aqueous liquid automatic dishwshing composition VII Liquidnonionic surfactant (e.g. alcohol  2.0-10.0% ethoxylates) Alkali metalsilicate  3.0-15.0% Alkali metal phosphate 20.0-40.0% Liquid carrierselected from higher 25.0-45.0% glycols, polyglycols, polyoxides,glycolethers Stabilizer (e.g. a partial ester of 0.5-7.0% phosphoricacid and a C₁₆-C₁₈ alkanol) Foam suppressor (e.g. silicone)   0-1.5%Enzymes 0.0001-0.1%   Non-aqueous liquid dishwashing composition VIIILiquid nonionic surfactant (e.g. alcohol  2.0-10.0% ethoxylates) Sodiumsilicate  3.0-15.0% Alkali metal carbonate  7.0-20.0% Sodium citrate0.0-1.5% Stabilizing system (e.g. mixtures of 0.5-7.0% finely dividedsilicone and low molecular weight dialkyl polyglycol ethers) Lowmolecule weight polyacrylate polymer  5.0-15.0% Clay gel thickener (e.g.bentonite)  0.0-10.0% Hydroxypropyl cellulose polymer 0.0-0.6% Enzymes0.0001-0.1%   Liquid carrier selected from higher Balance lycols,polyglycols, polyoxides and glycol ethers

[0528] Thixotropic liquid automatic dishwashing composition IX C₁₂-C₁₄fatty acid   0-0.5% Block co-polymer surfactant  1.5-15.0% Sodiumcitrate  0-12% Sodium tripolyphosphate  0-15% Sodium carbonate 0-8%Aluminium tristearate   0-0.1% Sodium cumene sulphonate   0-1.7%Polyacrylate thickener 1.32-2.5%  Sodium polyacrylate 2.4-6.0% Boricacid   0-4.0% Sodium formate   0-0.45% Calcium formate   0-0.2% Sodiumn-decydiphenyl oxide disulphonate   0-4.0% Monoethanol amine (MEA)  0-1.86% Sodium hydroxide (50%) 1.9-9.3% 1,2-Propanediol   0-9.4%Enzymes 0.0001-0.1%   Suds suppressor, dye, perfumes, water Balance

[0529] Liquid automatic dishwashing composition X Alcohol ethoxylate0-20% Fatty acid ester sulphonate 0-30% Sodium dodecyl sulphate 0-20%Alkyl polyglycoside 0-21% Oleic acid 0-10% Sodium disilicate monohydrate18-33%  Sodium citrate dihydrate 18-33%  Sodium stearate  0-2.5% Sodiumperborate monohydrate 0-13% Tetraacetyl ethylene diamine (TAED) 0-8% Maleic acid/acrylic acid copolymer 4-8%  Enzymes 0.0001-0.1%   Liquidautomatic dishwashing composition containing protected bleach particlesXI Sodium silicate 5-10% Tetrapotassium pyrophosphate 15-25%  Sodiumtriphosphate 0-2%  Potassium carbonate 4-8%  Protected bleach particles,e.g. chlorine 5-10% Polymeric thickener 0.7-1.5%  Potassium hydroxide0-2%  Enzymes 0.0001-0.1%   Water Balance

[0530] XII: Automatic dishwashing compositions as described in I, II,III, IV, VI and X, wherein perborate is replaced by percarbonate.

[0531] XIII: Automatic dishwashing compositions as described in I-VI,which additionally contain a manganese catalyst. The manganese catalystmay, e.g., be one of the compounds described in “Efficient manganesecatalysts for low-temperature bleaching”, Nature, (1994), 369, 637-639.

[0532] Materials and Methods

[0533] Textiles:

[0534] WFK10N standard textile pieces (egg stains) were obtained fromWFK Testgewebe GmbH, Christenfeld 10, D-41379 Brüggen-Bracht, Germany.

[0535] Strains:

[0536]B. subtilis DN1885 (Diderichsen et al., 1990).

[0537]B. lentus 309 and 147 are specific strains of Bacillus lentus,deposited with the NCIB and accorded the accession numbers NCIB 10309and 10147, and described in U.S. Pat. No. 3,723,250 incorporated byreference herein.

[0538]E. coli MC 1000 (M. J. Casadaban and S. N. Cohen (1980); J. Mol.Biol. 138 179-207), was made r⁻,m⁺ by conventional methods and is alsodescribed in U.S. patent application Ser. No. 039,298.

[0539] Plasmids:

[0540] pJS3: E. coli—B. subtilis shuttle vector containing a syntheticgene encoding for subtilase 309 (Described by Jacob Schiødt et al. inProtein and Peptide letters 3:39-44 (1996)).

[0541] pSX222: B. subtilis expression vector (described in WO 96/34946).

[0542] General Molecular Biology Methods:

[0543] Unless otherwise mentioned the DNA manipulations andtransformations were performed using standard methods of molecularbiology (Sambrook et al. (1989) Molecular cloning: A laboratory manual,Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al.(eds.) “Current protocols in Molecular Biology”. John Wiley and Sons,1995; Harwood, C. R., and Cutting, S. M. (eds.) “Molecular BiologicalMethods for Bacillus”. John Wiley and Sons, 1990).

[0544] Enzymes for DNA manipulations were used according to thespecifications of the suppliers.

[0545] Enzymes For DNA Manipulations

[0546] Unless otherwise mentioned all enzymes for DNA manipulations,such as e.g. restiction endonucleases, ligases etc., are obtained fromNew England Biolabs, Inc.

[0547] Proteolytic Activity

[0548] In the context of this invention proteolytic activity isexpressed in Kilo NOVO Protease Units (KNPU). The activity is determinedrelatively to an enzyme standard (SAVINASE®), and the determination isbased on the digestion of a dimethyl casein (DMC) solution by theproteolytic enzyme at standard conditions, i.e. 50° C., pH 8.3, 9 min.reaction time, 3 min. measuring time. A folder AF 220/1 is availableupon request to Novozymes A/S, Denmark, which folder is hereby includedby reference.

[0549] A GU is a Glycine Unit, defined as the proteolytic enzymeactivity which, under standard conditions, during a 15 minutes'incubation at 40° C., with N-acetyl casein as substrate, produces anamount of NH₂-group equivalent to 1 mmole of glycine.

[0550] Enzyme activity can also be measured using the PNA assay,according to reaction with the soluble substratesuccinyl-alanine-alanine-proline-phenyl-alanine-para-nitro-phenol, whichis described in the Journal of American Oil Chemists Society, Rothgeb,T. M., Goodlander, B. D., Garrison, P. H., and Smith, L. A., (1988).

[0551] Fermentation:

[0552] Fermentations for the production of subtilase enzymes wereperformed at 30° C. on a rotary shaking table (300 r.p.m.) in 500 mlbaffled Erlenmeyer flasks containing 100 ml BPX medium for 5 days.

[0553] Consequently in order to make an e.g. 2 liter broth 20 Erlenmeyerflasks were fermented simultaneously.

[0554] Media: BPX Medium Composition (per liter) Potato starch 100 gGround barley 50 g Soybean flour 20 g Na₂HPO₄ × 12 H₂O 9 g Pluronic 0.1g Sodium caseinate 10 g

[0555] The starch in the medium is liquefied with α-amylase and themedium is sterilized by heating at 120° C. for 45 minutes. Aftersterilization the pH of the medium is adjusted to 9 by addition ofNaHCO₃ to 0.1 M.

EXAMPLE 1

[0556] Construction and Expression of Enzyme Variants:

[0557] Site-Directed Mutagenesis:

[0558] Subtilase 309 (SAVINASE®) site-directed variants of the inventioncomprising specific insertions and comprising specific substitutionswere made by traditional cloning of DNA fragments (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989) produced by PCR with oligos containing the desired insertions (seebelow).

[0559] The template plasmid DNA was pJS3 (see below), or an analogue ofthis containing a variant of Subtilase 309.

[0560] Insertions and substitutions were introduced by oligo directedmutagenesis to the construction of variants.

[0561] The Subtilase 309 variants were transformed into E. coli. DNApurified from a over night culture of these transformants weretransformed into B. subtilis by restriction endonuclease digestion,purification of DNA fragments, ligation, transformation of B. subtilis.Transformation of B. subtilis was performed as described by Dubnau etal., 1971, J. Mol. Biol. 56, pp. 209-221.

[0562] Site-Directed Mutagenesis in Order to Introduce Insertions andSubstitutions in a Specific Region:

[0563] The overall strategy to used to perform site-directed mutagenesiswas:

[0564] Mutagenic primers (oligonucleotides) were synthesizedcorresponding to the DNA sequence flanking the sites of insertion andsubstitutions, separated by the DNA base pairs defining the insertionsand substitutions.

[0565] Subsequently, the resulting mutagenic primers were used in a PCRreaction with the modified plasmid pJS3 (see above). The resulting PCRfragment was purified and extended in a second PCR-reaction, theresulting PCR product was purified and extended in a third PCR-reactionbefore being digested by endonucleases and cloned into the E. coli—B.subtilis shuttle vector (see below). The PCR reactions are performedunder normal conditions.

[0566] Following this strategy two insertion and one substitution wasconstructed in SAVINASE® wherein insertions was introduced in position99 (*99aD) and 217 (*217aP) respectively and a substitution wasintroduced in position S99A (see below).

[0567] The insertion and substitution at position 99 was introduced by amutagenic primer (5′ CCG AAC CTG AAC CAT CCG CGG CCC CTA GGA CTT TAA CAGC 3′ (sense) (SEQ ID NO: 3) )were used in a PCR reaction with anopposite primer (5′ GAG TTA AGC CCA GAA GAT GTG GAC GCG 3′ (antisense)(SEQ ID NO: 4)).

[0568] The produced PCR fragment were extended towards the C-terminal ofSAVINASE by a second round of PCR introducing the insertion at position217 with primer 5′ CAT CGA TGT ACC GTT TGG TAA GCT GGC ATA TGT TG 3′(SEQ ID NO: 5). The second round PCR product were extended towards theC-terminal of SAVINASE by a third round of PCR with primer; 5′ AAC CGCACA GCG TTT TTT TAT TGA TTA ACG CGT TGC 3′ (SEQ ID NO: 6), situateddownstream at the Mlu I site in pJS3. All PCR reactions used plasmidpJS3 as template. The extended DNA-fragment resulting from third roundPCR was cloned into the Sal I- and Mlu I-sites of the modified plasmidpJS3 (see above).

[0569] The plasmid DNA was transformed into E. coli by well-knowntechniques and one E. coli colony were sequenced to confirm the mutationdesigned.

[0570] In order to purify a subtilase variant of the invention, the B.subtilis pJS3 expression plasmid comprising a variant of the inventionwas transformed into a competent B. subtilis strain and was fermented asdescribed above in a medium containing 10 μg/ml Chloramphenicol (CAM).

EXAMPLE 2

[0571] Purification of Enzyme Variants:

[0572] This procedure relates to purification of a 2 liter scalefermentation for the production of the subtilases of the invention in aBacillus host cell.

[0573] Approximately 1.6 liters of fermentation broth were centrifugedat 5000 rpm for 35 minutes in 1 liter beakers. The supernatants wereadjusted to pH 6.5 using 10% acetic acid and filtered on Seitz SupraS100 filter plates.

[0574] The filtrates were concentrated to approximately 400 ml using anAmicon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UFconcentrate was centrifuged and filtered prior to absorption at roomtemperature on a Bacitracin affinity column at pH 7. The protease waseluted from the Bacitracin column at room temperature using 25%2-propanol and 1 M sodium chloride in a buffer solution with 0.01dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chlorideadjusted to pH 7.

[0575] The fractions with protease activity from the Bacitracinpurification step were combined and applied to a 750 ml Sephadex G25column (5 cm dia.) equilibrated with a buffer containing 0.01dimethylglutaric acid, 0.2 M boric acid and 0.002 M calcium chlorideadjusted to pH 6.5.

[0576] Fractions with proteolytic activity from the Sephadex G25 columnwere combined and applied to a 150 ml CM Sepharose CL 6B cation exchangecolumn (5 cm dia.) equilibrated with a buffer containing 0.01 Mdimethylglutaric acid, 0.2 M boric acid, and 0.002 M calcium chlorideadjusted to pH 6.5.

[0577] The protease was eluted using a linear gradient of 0-0.1 M sodiumchloride in 2 liters of the same buffer (0-0.2 M sodium chloride in caseof Subtilisin 147).

[0578] In a final purification step protease containing fractions fromthe CM Sepharose column were combined and concentrated in an Amiconultrafiltration cell equipped with a GR81PP membrane (from the DanishSugar Factories Inc.).

[0579] By using the techniques of Example 1 for the construction andfermentation, and the above isolation procedure the following subtilisin309 variants were produced and isolated:

[0580] L42LN+P129PA

[0581] S99SD+L42LN

[0582] L42LN+L217LP

[0583] L42LN+S99SD+P129PA

[0584] S99A+S99SD+N155NA=S99AD+N155NA

[0585] S99A+S99SD+N155ND=S99AD+N155ND

[0586] S99A+S99SD+N155NR=S99AD+N155NR

[0587] S99A+S99SD+N155NF=S99AD+N155NF

[0588] S99A+S99SD+S188SE=S99AD+S188SE

[0589] S99A+S99SD+S188SD=S99AD+S188SD

[0590] S99A+S99SD+S188SK=S99AD+S188SK

[0591] S99A+S99SD+S188SL=S99AD+S188SL

[0592] S99A+S99SD+S188SA=S99AD+S188SA

[0593] S99A+S99SD+S216SP=S99AD+S216SP

[0594] S99A+S99SD+S216SDP=S99AD+S216SDP

[0595] S99A+S99SD+S216SPD=S99AD+S216SPD

[0596] S99D+S101R+S103A+V104I+G160S+A194P+V199M+V205I+S216SD

[0597] S99D+S101R+S103A+V104I+G160S+A194P+V199M+V205I+S216SE

[0598] L217LP

[0599] L217LA

[0600] L217LDP

[0601] S99SD+L217LP

[0602] P129PA+L217LP

[0603] S99A+S99SD+L217LP=S99AD+L217LP

[0604] S99A+S99SD+L217LDP=S99AD+L217LDP

[0605] S99A+S99SD+L217LPD=S99AD+L217LPD

[0606] S99D+S101R+S103A+V104I+G160S+A194P+V199M+V205I+L217LD

[0607] S99D+S101R+S103A+V104I+G160S+A194P+V199M+V205I+L217LE

[0608] S99A+S99SD+N218NP=S99AD+N218NP

[0609] S99A+S99SD+N218NDP=S99AD+N218NDP

[0610] S99A+S99SD+N218NPD=S99AD+N218NPD

EXAMPLE 3

[0611] The “Model Detergent Wash Performance Test”:

[0612] In order to asses the wash performance of selected subtilasevariants in a standard detergent composition, standard washingexperiments may be performed using the below experimental conditions:Detergent: Model detergent Detergent dosage 4.0 g/l pH 10.1 Wash time 20min Temperature: 30° C. Water hardness: 15° dH Enzyme concentration: 10nm (in the detergent solution) Test system: 10 ml beakers with astirring rod Textile/volume: 5 textile pieces ( 2.5 cm)/50 ml detergentsolution Test material: WFK10N (egg stains)

[0613] The composition of the model detergent is as follows:

[0614] 6.2% LAS (Nansa 80S)

[0615] 2% Sodium salt of C₁₆-C₁₈ fatty acid

[0616] 4% Non-ionic surfactant (Plurafax LF404)

[0617] 22% Zeolite P

[0618] 10.5% Na₂CO₃

[0619] 4% Na₂Si₂O₅

[0620] 2% Carboxymethylcellulose (CMC)

[0621] 6.8% Acrylate liquid CP5 40%

[0622] 20% Sodium perborate (empirical formula NaBO₂.H₂O₂)

[0623] 0.2% EDTA

[0624] 21% Na₂SO₄

[0625] Water (balance)

[0626] pH of the detergent solution is adjusted to 10.1 by addition ofHCl or NaOH. Water hardness is adjusted to 15° dH by addition of CaCl₂and MGCl₂ (Ca²⁺:Mg²⁺=4:1) to the test system. After washing the textilepieces are flushed in tap water and air-dried.

[0627] Measurement of the reflectance (R_(variant)) on the test materialis performed at 460 nm using a Macbeth ColorEye 7000 photometer(Macbeth, Division of Kollmorgen Instruments Corporation, Germany). Themeasurements are performed accordance with the manufacturer's protocol.

[0628] In order to determine a blank value, a similar wash experiment isperformed without addition of enzyme. The subsequent measurement of thereflectance (R_(blank)) is performed as described right above.

[0629] A reference experiment is then performed as described above,wherein the wash performance of the parent enzyme is tested. Thesubsequent measurement of the reflectance (R_(parent)) is performed asdescribed right above.

[0630] The wash performance is evaluated by means of the PerformanceFactor (P) which is defined in accordance with the below formula:$\begin{matrix}{P = {\left( {R_{variant} - R_{blank}} \right) - \left( {R_{parent} - R_{blank}} \right)}} \\{= {R_{variant} - {R_{parent}.}}}\end{matrix}$

EXAMPLE 4

[0631] The “Ovo-Inhibition Assay”

[0632] The below inhibition assay is based on the principle that thesubtilase variant to be tested will catalyse the hydrolysis of apeptide-pNA bond, thereby releasing the yellow pNA, which mayconveniently be followed at 405 nm. The amount of released pNA after agiven period of time is a direct measure of the subtilase activity. Bycarrying out such hydrolysis experiments with and without inhibitor,respectively, it is possible to obtain a quantitative measure for thedegree to which a certain subtilase variant is inhibited. Reactionconditions: Enzyme concentration: 0.0003 mg/ml Conc. of trypsininhibitor type IV-0: 0.0015 mg/ml Initial substrate concentration: 0.81mM Reaction time: 11 min Assay temperature: 25° C. Assay pH: 8.6Absorbance measured at: 405 nm

[0633] Assay Solutions:

[0634] Substrate Solution (2 mM):

[0635] 500 mg Suc-Ala-Ala-Pro-Phe-pNA is dissolved in 4 ml DMSO (200mM). This solution is diluted 100 times with the buffer solutiondescribed below. The concentration of substrate in the resultingsubstrate solution is 2 mM.

[0636] Inhibitor Solution (0.005 mg/ml):

[0637] 5 mg trypsin inhibitor type IV-0 (Sigma T-1886) is dissolved in10 ml water. This solution is dissolved 100 times with the buffersolution described below. The concentration of inhibitor in theresulting inhibitor solution is 0.005 mg/ml.

[0638] Enzyme Solution (0.001 mg/ml):

[0639] 1 mg enzyme is dissolved in 10 ml water. This solution isdissolved 100 times with the buffer solution described below. Theconcentration of enzyme in the resulting enzyme solution is 0.001 mg/ml.

[0640] Buffer Solution (pH 8.6):

[0641] 15.7 mg Tris is dissolved in an appropriate amount of water and0.75 ml 30% (w/v) BRIJ (BRIJ 35 polyoxyethylenelaurylether, 30% (w/v),Sigma Cat. No. 430AG-6) is added. The pH is adjusted to 8.6 with 4 MNaOH and the solution is diluted to 1 liter with water.

[0642] Assay with Inhibitor

[0643] 1 volume unit (e.g. 80 μl) inhibitor solution is mixed with 1volume unit (e.g. 80 μl) enzyme solution in an appropriate reactionvessel (e.g. a spectrophotometer cell or a micro titer plate) andequilibrated at 25° C. for 15 min. 1.375 volume units (e.g. 110 μl)substrate solution is added to the reaction vessel after which theabsorbance at 405 nm is followed for 11 min (e.g. by measuring every10^(th) or 30^(th) second). The slope of the absorbance curve iscalculated using linear regression analysis. The slope of the absorbancecurve is denoted α_(inhibitor).

[0644] Assay without Inhibitor

[0645] 1 volume unit (e.g. 80 μl) buffer solution is mixed with 1 volumeunit (e.g. 80 μl) enzyme solution in an appropriate reaction vessel(e.g. a spectrophotometer cell or a micro titer plate) and equilibratedat 25° C. for 15 min. 1.375 volume units (e.g. 110 μl) substratesolution is added to the reaction vessel after which the absorbance at405 nm is followed for 11 min (e.g. by measuring every 10^(th) or30^(th) second). The slope of the absorbance curve is calculated usinglinear regression analysis. The slope of the absorbance curve is denotedα.

[0646] Blank

[0647] 1 volume unit (e.g. 80 μl) inhibitor solution is mixed with 1volume unit (e.g. 80 μl) buffer solution in an appropriate reactionvessel (e.g. a spectrophotometer cell or a micro titer plate) andequilibrated at 25° C. for 15 min. 1.375 volume units (e.g. 110 μl)substrate solution is added to the reaction vessel after which theabsorbance at 405 nm is followed for 15 min. These measurements are notused in the calculations, but merely serve as a control that no enzymehas been added to the buffer and/or substrate solution.

[0648] Calculation of Residual Activity (RA)

[0649] The residual enzyme activity (RA) is calculated according to thebelow formula:

RA=(α_(inhibitor)/α)×100%

[0650] Using the above test, the following results were obtained:Residual Activity Enzyme (%) SAVINASE ®   <5% L217LP + S99SD + S99A97.0% S99D + S101R + S103A + V104I + G160S + A194P + V199M + V205I +*216aD 24.0% S99D + S101R + S103A + V104I + G160S + A194P + V199M +V205I + *216aE 25.4% S99SD + S99A + S216SDP 35.0% S99SD + S99A + N155NA 7.4% S99SD + S99A + N155ND 10.7% S99SD + S99A + N155NR  6.5% S99SD +S99A + N155ND  7.1% L42LN 69.2%

[0651]

1 8 1 5 PRT Artificial Sequence Synthetic 1 Ala Gly Lys Ala Leu 1 5 2 4PRT Artificial Sequence Synthetic 2 Ala Gly Gly Leu 1 3 40 DNAArtificial Sequence Primer 3 ccgaacctga accatccgcg gcccctagga ctttaacagc40 4 27 DNA Artificial Sequence Primer 4 gagttaagcc cagaagatgt ggacgcg27 5 35 DNA Artificial Sequence Primer 5 catcgatgta ccgtttggtaagctggcata tgttg 35 6 36 DNA Artificial Sequence Primer 6 aaccgcacagcgttttttta ttgattaacg cgttgc 36 7 275 PRT Bacillus 7 Ala Gln Ser Val ProTyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln GlyTyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile AspSer Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala 35 40 45 Ser Met Val ProSer Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 50 55 60 Gly Thr His ValAla Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu GlyVal Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Gly Ala AspGly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp AlaIle Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 ProSer Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala 130 135 140Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly 145 150155 160 Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala165 170 175 Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser SerVal 180 185 190 Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile GlnSer Thr 195 200 205 Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr SerMet Ala Ser 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu SerLys His Pro Asn 225 230 235 240 Trp Thr Asn Thr Gln Val Arg Ser Ser LeuGlu Asn Thr Thr Thr Lys 245 250 255 Leu Gly Asp Ser Phe Tyr Tyr Gly LysGly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 8 269 PRTBacillus 8 Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro AlaAla 1 5 10 15 His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala ValLeu Asp 20 25 30 Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly GlyAla Ser 35 40 45 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly HisGly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile GlyVal Leu 65 70 75 80 Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys ValLeu Gly Ala 85 90 95 Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly LeuGlu Trp Ala 100 105 110 Gly Asn Asn Gly Met His Val Ala Asn Leu Ser LeuGly Ser Pro Ser 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala Val Asn SerAla Thr Ser Arg Gly 130 135 140 Val Leu Val Val Ala Ala Ser Gly Asn SerGly Ala Gly Ser Ile Ser 145 150 155 160 Tyr Pro Ala Arg Tyr Ala Asn AlaMet Ala Val Gly Ala Thr Asp Gln 165 170 175 Asn Asn Asn Arg Ala Ser PheSer Gln Tyr Gly Ala Gly Leu Asp Ile 180 185 190 Val Ala Pro Gly Val AsnVal Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195 200 205 Ala Ser Leu Asn GlyThr Ser Met Ala Thr Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu ValLys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 225 230 235 240 Arg AsnHis Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245 250 255 TyrGly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265

1. A subtilase variant comprising an insertion of at least one aminoacid residue between: (a) positions 42 and 43; (b) positions 51 and 56;(c) positions 155 and 161; (d) positions 187 and 190; (e) positions 216and 217; (f) positions 217 and 218; and/or (g) positions 218 and 219;wherein the positions are numbered according to the amino acid sequenceof the mature subtilisin BPN′.
 2. The variant of claim 1, wherein theinsertion is between positions 51 and 52; positions 52 and 53; positions53 and 54; positions 54 and 55; or positions 55 and
 56. 3. The variantof claim 1, wherein the insertion is between positions 155 and 156;positions 156 and 157; positions 157 and 158; positions 158 and 159;positions 159 and 160; or positions 160 and
 161. 4. The variant of claim1, wherein the insertion is between positions 187 and 188; positions 188and 189; or positions 189 and
 190. 5. The variant of claim 1, where thevariant has a residual activity of at least 10% in the Ovo-inhibitionassay.
 6. The variant of claim 5, where the variant has a residualactivity of at least 15%, preferably at least 20%, more preferably atleast 25%.
 7. The variant of claim 1, wherein the variant contains morethan one insertion between said positions.
 8. The variant of claim 1,wherein the variant contains two or more insertions between differentsets of the above positions.
 9. The variant of claim 8, wherein thevariant contains more than two insertions between each of said sets ofpositions.
 10. The variant of claim 1, wherein the insertion is selectedfrom the group consisting of X42XA, X42XT, X42XG, X42XS, X42XD, X42XE,X42XK, X42XR, X42XH, X42XV, X42XC, X42XN, X42XQ, X42XF, X42XI, X42XL,X42XM, X42XP, X42XW and X42XY.
 11. The variant of claim 2, wherein theinsertion is selected from the group consisting of X51XA, X51XT, X51XG,X51XS, X51XD, X51XE, X51XK, X51XR, X51XH, X51XV, X51XC, X51XN, X51XQ,X51XF, X51XI, X51XL, X51XM, X51XP, X51XW and X51XY.
 12. The variant ofclaim 2, wherein the insertion is selected from the group consisting ofX52XA, X52XT, X52XG, X52XS, X52XD, X52XE, X52XK, X52XR, X52XH, X52XV,X52XC, X52XN, X52XQ, X52XF, X52XI, X52XL, X52XM, X52XP, X52XW and X52XY.13. The variant of claim 2, wherein the insertion is selected from thegroup consisting of X53XA, X53XT, X53XG, X53XS, X53XD, X53XE, X53XK,X53XR, X53XH, X53XV, X53XC, X53XN, X53XQ, X53XF, X53XI, X53XL, X53XM,X53XP, X53XW and X53XY.
 14. The variant of claim 2, wherein theinsertion is selected from the group consisting of X54XA, X54XT, X54XG,X54XS, X54XD, X54XE, X54XK, X54XR, X54XH, X54XV, X54XC, X54XN, X54XQ,X54XF, X54XI, X54XL, X54XM, X54XP, X54XW and X54XY.
 15. The variant ofclaim 2, wherein the insertion is selected from the group consisting ofX55XA, X55XT, X55XG, X55XS, X55XD, X55XE, X55XK, X55XR, X55XH, X55XV,X55XC, X55XN, X55XQ, X55XF, X55XI, X55XL, X55XM, X55XP, X55XW and X55XY.16. The variant of claim 3, wherein the insertion is selected from thegroup consisting of X155XA, X155XT, X155XG, X155XS, X155XD, X155XE,X155XK, X155XR, X155XH, X155XV, X155XC, X155XN, X155XQ, X155XF, X155XI,X155XL, X155XM, X155XP, X155XW and X155XY.
 17. The variant of claim 3,wherein the insertion is selected from the group consisting of X156XA,X156XT, X156XG, X156XS, X156XD, X156XE, X156XK, X156XR, X156XH, X156XV,X156XC, X156XN, X156XQ, X156XF, X156XI, X156XL, X156XM, X156XP, X156XWand X156XY.
 18. The variant of claim 3, wherein the insertion isselected from the group consisting of X157XA, X157XT, X157XG, X157XS,X157XD, X157XE, X157XK, X157XR, X157XH, X157XV, X157XC, X157XN, X157XQ,X157XF, X157XI, X157XL, X157XM, X157XP, X157XW and X157XY.
 19. Thevariant of claim 3, wherein the insertion is selected from the groupconsisting of X158XA, X158XT, X158XG, X158XS, X158XD, X158XE, X158XK,X158XR, X158XH, X158XV, X158XC, X158XN, X158XQ, X158XF, X158XI, X158XL,X158XM, X158XP, X158XW and X158XY.
 20. The variant of claim 3, whereinthe insertion is selected from the group consisting of X159XA, X159XT,X159XG, X159XS, X159XD, X159XE, X159XK, X159XR, X159XH, X159XV, X159XC,X159XN, X159XQ, X159XF, X159XI, X159XL, X159XM, X159XP, X159XW andX159XY.
 21. The variant of claim 3, wherein the insertion is selectedfrom the group consisting of X160XA, X160XT, X160XG, X160XS, X160XD,X160XE, X160XK, X160XR, X160XH, X160XV, X160XC, X160XN, X160XQ, X160XF,X160XI, X160XL, X160XM, X160XP, X160XW and X160XY.
 22. The variant ofclaim 4, wherein the insertion is selected from the group consisting ofX187XA, X187XT, X187XG, X187XS, X187XD, X187XE, X187XK, X187XR, X187XH,X187XV, X187XC, X187XN, X187XQ, X187XF, X187XI, X187XL, X187XM, X187XP,X187XW and X187XY.
 23. The variant of claim 4, wherein the insertion isselected from the group consisting of X188XA, X188XT, X188XG, X188XS,X188XD, X188XE, X188XK, X188XR, X188XH, X188XV, X188XC, X188XN, X188XQ,X188XF, X188XI, X188XL, X188XM, X188XP, X188XW and X188XY.
 24. Thevariant of claim 4, wherein the insertion is selected from the groupconsisting of X189XA, X189XT, X189XG, X189XS, X189XD, X189XE, X189XK,X189XR, X189XH, X189XV, X189XC, X189XN, X189XQ, X189XF, X189XI, X189XL,X189XM, X189XP, X189XW and X189XY.
 25. The variant of claim 1, whereinthe insertion is selected from the group consisting of X216XA, X216XT,X216XG, X216XS, X216XD, X216XE, X216XK, X216XR, X216XH, X216XV, X216XC,X216XN, X216XQ, X216XF, X216XI, X216XL, X216XM, X216XP, X216XW andX216XY.
 26. The variant of claim 1, wherein the insertion is selectedfrom the group consisting of X217XA, X217XT, X217XG, X217XS, X217XD,X217XE, X217XK, X217XR, X217XH, X217XV, X217XC, X217XN, X217XQ, X217XF,X217XI, X217XL, X217XM, X217XP, X217XW and X217XY.
 27. The variant ofclaim 1, wherein the insertion is selected from the group consisting ofX218XA, X218XT, X218XG, X218XS, X218XD, X218XE, X218XK, X218XR, X218XH,X218XV, X218XC, X218XN, X218XQ, X218XF, X218XI, X218XL, X218XM, X218XP,X218XW and X218XY.
 28. The variant of claim 1, wherein the parentsubtilase belongs to the sub-group I-S1.
 29. The variant of claim 28,wherein the parent subtilase is selected from the group consisting ofBSS168, BSSAS, BSAPRJ, BSAPRN, BMSAMP, BASBPN, BSSDY, BLSCAR BLKERA,BLSCA1, BLSCA2, BLSCA3, BSSPRC, and BSSPRD, or functional variantsthereof having retained the characteristics of sub-group I-S1.
 30. Thevariant of claim 1, wherein the parent subtilase belongs to thesub-group I-S2.
 31. The variant of claim 30, wherein the parentsubtilase is selected from the group consisting of BSAPRQ, BLS147BSAPRM, BAH101, BLSAVI (BLS309), BSKSMK, BAALKP (BAPB92) BLSUBL, BSEYAB,TVTHER, and BSAPRS, or functional variants thereof having retained thecharacteristics of sub-group I-S2.
 32. The variant of claim 31, whereinthe parent subtilase is BLSAVI.
 33. The variant of claim 1, wherein thevariant comprises at least one further modification.
 34. The variant ofclaim 33, wherein the modification is a substitution.
 35. The variant ofclaim 1, wherein the variant further comprises at least one modificationin position 27, 36, 56, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 120, 123, 129, 131, 132, 133, 143, 159, 167, 170, 192, 194, 206,217, 218, 222, 224, 232, 235, 236, 245, 248, 252 or
 274. 36. The variantof claim 35, wherein the variant further comprises the modificationS101G+S103A+V104I+G159D+A232V+Q236H+Q245R+N248D+N252K.
 37. The variantof claim 1, wherein the variant further comprises at least oneadditional amino acid residue in the active site loop (b) region fromposition 95 to
 103. 38. A cleaning or detergent composition, comprisingthe variant of claim 1 and a surfactant.
 39. The composition of claim38, which additionally comprises an amylase, cellulase, cutinase,lipase, oxidoreductase, another protease, or combination thereof.
 40. Amethod for removal of egg stains from a hard surface or from laundry,comprising contacting the egg stain-containing hard surface or the eggstain-containing laundry with a cleaning or detergent composition ofclaim
 38. 41. The method of claim 40, wherein the compositionadditionally comprises an amylase, cellulase, cutinase, lipase,oxidoreductase, another protease, or combination thereof.
 42. Anisolated polynucleotide with a DNA sequence encoding a subtilase variantof claim
 1. 43. An expression vector comprising the isolatedpolynucleotide of claim
 42. 44. A microbial host cell transformed withthe expression vector of claim
 43. 45. A microbial host cell of claim44, which is a bacterium, preferably a Bacillus, especially a B. lentus.46. A microbial host cell of claim 44, which is a fungus or yeast,preferably a filamentous fungus, especially an Aspergillus.
 47. A methodfor producing a subtilase variant, comprising culturing a host of claim44 under conditions conducive to the expression and secretion of thevariant, and recovering the variant.